Instruments, modules, and methods for improved detection of edited sequences in live cells

ABSTRACT

The present disclosure provides instruments, modules and methods for improved detection of edited cells following nucleic acid-guided nuclease genome editing. The disclosure provides improved automated instruments that perform methods—including high throughput methods—for screening cells that have been subjected to editing and identifying cells that have been properly edited.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/597,826, filed 9Oct. 2020, now U.S. Pat. No. 10,633,626; which is a continuation of U.S.Ser. No. 16/454,865, filed 27 Jun. 2019, now U.S. Pat. No. 10,550,363;which is a continuation of U.S. Ser. No. 16/399,988, filed 30 Apr. 2019,now U.S. Pat. No. 10,533,152; which claims priority to U.S. ProvisionalApplications No. 62/718,449, filed 14 Aug. 2018; 62/735,365, filed 24Sep. 2018; 62/781,112, filed 18 Dec. 2018; and 62/779,119, filed 13 Dec.2018.

FIELD OF THE INVENTION

This invention relates to improved instruments, modules, and methods toscreen, select and thus optimize detection of genome edits in livecells.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that themethods referenced herein do not constitute prior art under theapplicable statutory provisions.

The ability to make precise, targeted changes to the genome of livingcells has been a long-standing goal in biomedical research anddevelopment. Recently, various nucleases have been identified that allowfor manipulation of gene sequences, and hence gene function. Thenucleases include nucleic acid-guided nucleases, which enableresearchers to generate permanent edits in live cells. Current protocolsemploying nucleic acid-guided nuclease systems typically utilizeconstitutively-expressed nuclease components to drive high efficiencyediting. However, in pooled or multiplex formats constitutive expressionof editing components can lead to rapid depletion of edited cell typesand selective enrichment of cells that have not been edited. This occursin most cell types because only a small fraction (<1-5%) of cellssurvive the introduction of double-strand DNA (dsDNA) breaks and thusthese cells contribute fewer numbers to viable cell counts in theresulting populations compared to unedited cells that did not experiencea dsDNA break.

There is thus a need in the art of nucleic acid-guided nuclease geneediting for improved instruments, modules and methods for creatinggenome edits and for identifying and enriching cells that have beenedited. The present invention satisfies this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides instruments, modules and methods fortightly-regulated expression of nucleic acid-guided nuclease editingsystem components that allow for separation of the processes oftransformation and genome editing. The compositions and methods employinducible guide RNA (gRNA) constructs leading to increased observedtransformation efficiency and automation-friendly control over thetiming and duration of the editing process. Further, the instruments,modules, and methods enable automated high-throughput and extremelysensitive screening to identify edited cells. The instruments, modules,and methods take advantage of singulation or substantial singulation,where the term “singulation” in this context refers to the process ofseparating cells and growing them into clonally-isolated formats. Theterm “substantial singulation” refers to the process of separating cellsin a population of cells into “groups” of 2 to 100, or 2 to 50, andpreferably 2 to 10 cells. Singulation, followed by an initial period ofgrowth, induction of editing, and growth normalization leads toenrichment of edited cells. Further, the instruments, modules, andmethods described herein facilitate “cherry picking” of edited cellcolonies, allowing for direct selection of edited cells. Singulation orsubstantial singulation assists in overcoming the growth bias fromunedited cells that occurs under competitive growth regimes such as inbulk liquid culture. Indeed, it has been determined that removing growthrate bias via singulation or substantial singulation, induction andnormalization improves the observed editing efficiency by up to 4×(from, e.g., 10% to 40% absolute efficiency at population scale) or moreover conventional methods, and further that cherry-picking coloniesusing the methods described herein brings the observed editingefficiency up to 8× (from, e.g., 10% to 80% absolute efficiency atpopulation scale) over conventional methods. Thus, the combination ofsingulation or substantial singulation, growth, induction of editing,and either normalization or cherry picking improves observed editingefficiency by up to 8× over conventional methods where singulation orsubstantial singulation, growth, induction of editing and eithernormalization or cherry picking are not employed.

One particularly facile module or device for singulation or substantialsingulation is a solid wall device where cells are substantiallyisolated, grown in a clonal format, editing is induced, and eithernormalization or cherry picking is employed. The solid wall devices ormodules and the use thereof are described in detail herein. Theinstruments, modules and methods in some embodiments allow fornormalization of edited and unedited cell colonies. Normalization refersto growing colonies of cells—whether edited or unedited—to terminalsize; that is, growing the cells until the cells in the colonies entersenescence due to, e.g., nutrient exhaustion or constrained space forfurther growth. Normalization of cell colonies enriches for edited cellsas edited cells get “equal billing” with unedited cells. Additionally,the instruments, modules, and methods facilitate “cherry picking” ofcolonies. Cherry picking allows for direct selection of edited cells bytaking advantage of edit-induced growth delay in edited colonies. Cherrypicking colonies using the instruments, modules, and methods describedherein may more than double the observed editing efficiency as theresult of singulation or substantial singulation.

Certain embodiments of the instruments, modules, and methods provide forenriching for edited cells during nucleic acid-guided nuclease editing,where the methods comprise transforming cells with one or more vectorscomprising a promoter driving expression of a nuclease, a promoterdriving transcription of a guide nucleic acid, and a donor DNA sequence,wherein one or both of the promoters driving transcription of thenuclease or guide nucleic acid is an inducible promoter; diluting thetransformed cells to a cell concentration sufficient to substantiallysingulate the transformed cells on a substrate; growing thesubstantially singulated cells on the substrate; initiating editing byinducing the inducible promoter(s); and either 1) growing the cellcolonies resulting from the induced cells to colonies of terminal size(e.g., normalizing the cell colonies) and harvesting the normalized cellcolonies; or 2) monitoring the growth of cells colonies on the substratethen selecting slow-growing colonies.

A solid wall isolation, induction and normalization (SWIIN) modulecomprising: a retentate member comprising: an upper surface and a lowersurface and a first and second end, an upper portion of a serpentinechannel defined by raised areas on the lower surface of the retentatemember, wherein the upper portion of the serpentine channel traversesthe lower surface of the retentate member for about 50% to about 90% ofthe length and width of the lower surface of the retentate member; atleast one port fluidically connected to the upper portion of theserpentine channel; and a reservoir cover at the first end of theretentate member; a permeate member disposed under the retentate membercomprising: an upper surface and a lower surface and a first and secondend, a lower portion of a serpentine channel defined by raised areas onthe upper surface of the permeate member, wherein the lower portion ofthe serpentine channel traverses the upper surface of the permeatemember for about 50% to about 90% of the length and width of the uppersurface of the permeate member, and wherein the lower portion of theserpentine channel is configured to mate with the upper portion of theserpentine channel to form a mated serpentine channel; at least one portfluidically connected to the lower portion of the serpentine channel;and a first and second reservoir at the first end of the permeatemember, wherein the first reservoir is fluidically connected to the atleast one port in the retentate member and the second reservoir isfluidically connected to the at least one port in the permeate member; aperforated member comprising at least 25,000 perforations disposed underand adjacent to the retentate member; a filter disposed disposed underand adjacent to the perforated member and above and adjacent to thepermeate member; and a gasket disposed on top of the reservoir cover ofthe retentate member, wherein the gasket comprises a reservoir accessaperture and a pneumatic access aperture for each reservoir.

In some aspects of this embodiment, the permeate member furthercomprises ultrasonic tabs disposed on the raised areas on the uppersurface of the permeate member and at the first and second end of thepermeate member, the retentate member further comprises recesses for theultrasonic tabs disposed in the raised areas on the lower surface and atthe first and second end of the retentate member, the ultrasonic tabsare configured to mate with the recesses for the ultrasonic tabs, andthe permeate member, retentate member, the perforated member and thefilter are coupled together by ultrasonic welding. In other aspects ofthis embodiment, the permeate member, retentate member, the perforatedmember and the filter are coupled together by solvent bonding.

In some aspects, the first and second reservoirs are each fluidicallycoupled to a reservoir port into which fluids and/or cells flow fromoutside the SWIIN module into the first and second reservoirs and areeach pneumatically coupled to a pressure source

In some aspects of this embodiment, the SWIIN module further comprises athird and a fourth reservoir, wherein the third reservoir is 1)fluidically coupled to a second port in the retentate member, 2)fluidically coupled to a reservoir port into which fluids and/or cellsflow from outside the SWIIN module into the third reservoir, and 3)pneumatically coupled to a pressure source; and

wherein the fourth reservoir is 1) fluidically coupled to a second portin the permeate member, 2) fluidically coupled to a reservoir port intowhich fluids and/or cells flow from outside the SWIIN module into thefourth reservoir, and 3) pneumatically coupled to a pressure source.

In some aspects of this embodiment, the perforated member comprises atleast 100,000 perforations, or at least 200,000 perforations, or atleast 400,000 perforations.

In some aspects, the retentate member is fabricated from polycarbonate,cyclic olefin co-polymer, or poly(methyl methylacrylate).

In some aspects of the SWIIN module, a serpentine channel portion ofeach of the retentate and permeate members is from 75 mm to 350 mm inlength, from 50 mm to 250 mm in width, and from 2 mm to 15 mm inthickness, and from 150 mm to 250 mm in length, from 100 mm to 150 mm inwidth, and from 4 mm to 8 mm in thickness.

In some aspects, the volume of the mated serpentine channel is from 4 to40 mL, or from 6 mL to 30 mL, or from 10 mL to 20 mL.

In some aspects of the SWIIN module, there is a support on each end ofthe permeate member configured to elevate the permeate and retentatemembers above the at least one port in the in retentate member and theat least one port in the permeate member.

Certain embodiments of the SWIIN module further comprises imaging meansto detect cells growing in the wells, and in some aspects, the imagingmeans is a camera with means to backlight the serpentine channel portionof the SWIIN. In some aspects, the SWIIN module is part of a SWIINassembly comprising a heated cover, a heater, a fan, and athermoelectric control device.

Also provided herein is an automated multi-module cell editinginstrument comprising: a SWIIN module, a housing configured to house allof some of the modules; a receptacle configured to receive cells; one ormore receptacles configured to receive nucleic acids; a growth module; atransformation module configured to introduce the nucleic acids into thecells; and a processor configured to operate the automated multi-modulecell editing instrument based on user input and/or selection of apre-programmed script.

In some aspects of this embodiment, the SWIIN module also serves as arecovery module and a curing module. In some aspects, the automatedmulti-module cell editing instrument further comprises a cellconcentration module. In some aspects, the transformation modulecomprises a flow-through electroporation device.

Other embodiments provide a method for enriching edited cells duringnucleic acid-guided nuclease editing comprising: transforming cells withone or more vectors comprising a promoter driving transcription of acoding sequence for a nuclease, a promoter driving transcription of aguide nucleic acid, and a DNA donor sequence; diluting the transformedcells to a cell concentration to substantially isolate the transformedcells on a substrate; growing the cells and initiating editing byinducing the inducible promoter driving transcription of the guidenucleic acid; growing the induced cells into colonies; and selectinginduced cells from the substantially isolated colonies from thesubstrate, wherein the induced substantially isolated colonies areenriched for edited cells. In optional aspects of this method, the gRNAis under the control of an inducible promoter and 1) the cells areallowed to grow from 2-200 doublings after singulation, and 2) there isan inducing step after the growth step and prior to the editing step.

Other embodiments provide a method for enriching edited cells duringnucleic acid-guided nuclease editing comprising: transforming cells withone or more vectors comprising comprising a promoter drivingtranscription of a coding sequence for a nuclease, a promoter drivingtranscription of a guide nucleic acid, and a DNA donor sequence;diluting the transformed cells to a cell concentration to substantiallyisolate the transformed cells on a substrate; growing the cells andallowing the cells to edit; growing the cells to form colonies; andselecting small colonies from the substantially isolated colonies fromthe substrate, wherein the induced substantially isolated colonies areenriched for edited cells. In optional aspects of this method, the gRNAis under the control of an inducible promoter and 1) the cells areallowed to grow from 2-200 doublings after singulation, and 2) there isan inducing step after the growth step and prior to the editing step.

Other embodiments provide a method for enriching edited cells duringnucleic acid-guided nuclease editing comprising: transforming cells withone or more vectors comprising a promoter driving transcription of acoding sequence for a nuclease, a promoter driving transcription of aguide nucleic acid, and a DNA donor sequence; diluting the transformedcells to a cell concentration to substantially isolate the transformedcells on a first substrate; growing the cells and allowing the cells toedit; and growing the cells to form colonies of terminal size. In someaspects, the terminal-size cell colonies are pooled, and in someaspects, the terminal-size colonies are picked. In optional aspects ofthis method, the gRNA is under the control of an inducible promoterand 1) the cells are allowed to grow from 2-200 doublings aftersingulation, and 2) there is an inducing step after the growth step andprior to the editing step.

Thus in some embodiments there is provided a singulation assembly for asolid wall singulation or substantial singulation, growth, induction ofediting, and normalization or cherry-picking (“solid wallinsolation/induction/normalization module” or “SWIIN”) modulecomprising: a retentate member comprising an upper surface and a lowersurface, wherein the retentate member comprises at least one retentatedistribution channel which traverses the retentate member from its uppersurface to its lower surface and for most of the length of retentatemember; wherein the lower surface of the retentate member comprisesretentate ridges between which are retentate flow directors; wherein theretentate member further comprises one or more retentate member portsconfigured to supply cells and fluid to and remove cells and fluid fromthe retentate member; and wherein the retentate member ports arefluidically-connected to the retentate distribution channel andretentate flow directors; a perforated member with an upper surface anda lower surface, wherein the upper surface of the perforated member ispositioned beneath and adjacent to the lower surface of the retentatemember and wherein the perforated member comprises at least 25,000perforations; a filter with an upper surface and a lower surface,wherein the upper surface of the filter is positioned beneath andadjacent to the lower surface of the perforated member and wherein thelower surface of the filter is positioned above and adjacent to an uppersurface of a permeate member; a gasket surrounding the perforated memberand the filter; and the permeate member comprising the upper surface anda lower surface, wherein the permeate member comprises at least onepermeate distribution channel which traverses the permeate member fromits lower surface to its upper surface and for most of the length ofpermeate member; wherein the upper surface of the permeate membercomprises permeate ridges between which are permeate flow directors;wherein the permeate member further comprises one or more permeatemember ports configured to supply fluid to and remove fluid from thepermeate member; and wherein the permeate member ports arefluidically-connected to the permeate distribution channel and permeateflow directors; and means to couple the retentate member, perforatedmember, filter, gasket and permeate member.

In some aspects of the singulation assembly embodiment the means tocouple the retentate member, perforated member, filter, gasket andpermeate member comprises ultrasonic welding and in other aspects, themeans comprises pressure sensitive adhesive, solvent bonding, matedfittings, or a combination of adhesives, welding, solvent bonding, andmated fittings; and other such fasteners and couplings. In some aspectsof the singulation assembly, the perforated member comprises at least50,000; 100,000; 150,000; 200,000, 250,000 perforations or more, and insome aspects, the SWIIN is a compound SWIIN and each part of thecompound SWIIN comprises a perforated member with at least 50,000;100,000; 150,000; 200,000, 250,000 perforations. In some aspects, theretentate and permeate members are fabricated from polycarbonate, cyclicolefin co-polymer, or poly(methyl methylacrylate); and in some aspects,the retentate and permeate members are from 75 mm to 350 mm in length,from 50 mm to 250 mm in width, and from 2 mm to 15 mm in thickness. Insome aspects of the singulation assembly, the retentate and permeatemembers are from 150 mm to 250 mm in length, from 100 mm to 150 mm inwidth, and from 4 mm to 8 mm in thickness. In some aspects of thesingulation assembly, there are two permeate distribution channelsand/or two retentate distribution channels, and in some aspects theretentate distribution channels and or permeate distribution channelsare approximately 150 mm in length and 1 mm in width. In some aspects,the retentate and/or permeate ridges are approximately 0.5 mm in heightand 80 mm in length, and in some aspects, the retentate and/or permeateflow directors are approximately 5 mm across. In some aspects, thevolume of the singulation assembly is from 15 mL to 100 mL.

Some embodiments of the disclosure provide a SWIIN module comprising thesingulation assembly and further comprising: a reservoir assemblycomprising at least two reservoirs wherein a first reservoir is 1)fluidically-coupled to at least one reservoir port into which fluidsand/or cells flow from outside the SWIIN module into the firstreservoir, 2) fluidically-coupled to a reservoir/channel port from whichfluids and/or cells flow into the one or more retentate member ports;and 3) pneumatically-coupled to a pressure source; a second reservoiris 1) fluidically-coupled to at least one reservoir port into whichfluids flow from outside the SWIIN module into the second reservoir, 2)fluidically-coupled to a reservoir/channel port from which fluids flowinto the one or more permeate member ports; and 3) pneumatically-coupledto a pressure source; and a SWIIN cover. In some aspects of the SWIINmodule embodiment, the SWIIN module further comprises two additionalreservoirs wherein a first and third reservoir are 1)fluidically-coupled to at least two reservoir ports into which fluidsand/or cells flow from outside the SWIIN module into the first and thirdreservoirs, 2) fluidically-coupled to a reservoir/channel port fromwhich fluids and/or cells flow into the at least two retentate memberports; and 3) pneumatically-coupled to a pressure source; and a secondand fourth reservoir are 1) fluidically-coupled to at least tworeservoir ports into which fluids flow from outside the SWIIN moduleinto the second and fourth reservoirs, 2) fluidically-coupled to areservoir/channel port from which fluids flow into the at least twopermeate member ports; and 3) pneumatically-coupled to a pressuresource.

In some aspects of the SWIIN module embodiment, the SWIIN covercomprises a reservoir cover portion of the SWIIN cover, and wherein thereservoir cover portion comprises 1) at least two reservoir accessapertures, wherein the reservoir access apertures provide access to thereservoir ports, and 2) at least two pneumatic access apertures, whereinthe pneumatic access apertures provide access to the at least tworeservoirs and provide negative and positive pressure to the at leasttwo reservoirs. In some aspects the SWIIN module is configured tomonitor cell colony growth after inducing editing, and further comprisesmeans for cherry picking slow growing cell colonies. In some aspects ofthe SWIIN module, editing is induced by an inducible promoter that is atemperature inducible promoter, and temperature to induce transcriptionof the nuclease and/or guide nucleic acid is provided to the SWIINmodule by a Peltier device.

Other embodiments of the disclosure provide an automated multi-modulecell editing instrument comprising: the SWIIN module; a housingconfigured to house all of some of the modules; a receptacle configuredto receive cells; one or more receptacles configured to receive nucleicacids; a growth module; a transformation module configured to introducethe nucleic acids into the cells; and a processor configured to operatethe automated multi-module cell editing instrument based on user inputand/or selection of a pre-programmed script.

In some aspects of the automated multi-module cell editing instrument,the transformation module comprises a flow-through electroporationdevice; and in some aspects the automated multi-module cell editinginstrument further comprises a cell concentration module. In someaspects the cell concentration module is a tangential flow filtrationmodule. In some aspects a liquid handling system transfers liquidsbetween the modules. And in some aspects, the automated multi-modulecell processing system performs the processes of growing cells,concentrating and rendering the cells electrocompetent, transforming thecells with nucleic acid-guided nuclease editing components, singulatingthe transformed cells, inducing editing in the singulated cells, andgrowing and enriching the cells, all without human intervention.

In yet other embodiments, there is provided an automated multi-modulecell editing instrument comprising: the SWIIN module; a housingconfigured to house all of some of the modules; a receptacle configuredto receive cells; one or more receptacles configured to receive nucleicacids; a transformation module configured to introduce the nucleic acidsinto the cells; a cell concentration module; and a processor configuredto operate the automated multi-module cell editing instrument based onuser input and/or selection of a pre-programmed script. In some aspectsof the automated multi-module cell processing instrument, and a liquidhandling system to transfer reagents to and between the modules, and theSWIIN module. In some aspects the stand-alone, integrated, automatedmulti-module cell processing system comprises a growth module whichcomprising a rotating growth vial, a nucleic acid assembly module, or areagent cartridge. And in some aspects, the automated multi-module cellprocessing system performs the processes of growing cells, concentratingand rendering the cells electrocompetent, transforming the cells withnucleic acid-guided nuclease editing components, singulating thetransformed cells, inducing editing in the singulated cells, and growingand enriching the cells, all without human intervention. As with otherembodiments, this embodiment of an automated multi-module cell editinginstrument comprises a liquid handling system to transfer liquidsbetween the modules.

In yet an additional embodiment, there is provided a method forenriching edited cells during nucleic acid-guided nuclease editingcomprising: transforming cells with one or more vectors comprising aninducible promoter driving expression of a nuclease, an induciblepromoter driving transcription of a guide nucleic acid, and a DNA donorsequence; diluting the transformed cells to a cell concentration tosubstantially singulate the transformed cells on a substrate; growingthe cells on the substrate for 2-200 doublings; initiating editing byinducing the inducible promoter(s) driving transcription of the guidenucleic acid and nuclease; growing the induced cells into colonies; andselecting induced cells from the substantially singulated colonies fromthe substrate, wherein the induced substantially singulated colonies areenriched for edited cells.

In some aspects of this method embodiment, the inducible drivingtranscription the guide nucleic acid is a pL promoter, and the induciblepromoter driving expression of the nuclease is a pL promoter. In someaspects, all of the nuclease, guide nucleic acid and DNA donor sequenceare on the same vector. In yet other aspects, the nuclease is on a firstvector and the guide nucleic acid and DNA donor sequence are on a secondvector and two transforming steps are required. In some aspects, the DNAdonor sequence further comprises a PAM-altering sequence, and in someaspects, the one or more vectors each further comprise a gene for aselectable marker. In some aspects, the method further comprises addingselective agents to medium of the substrate to select for the selectablemarker(s) on the one or more vectors. In some aspects, the inducedsubstantially singulated colonies selected are small colonies, yet inother aspects, the cells are grown into colonies of terminal size.

Also presented in one method embodiment is a method for enriching editedcells during nucleic acid-guided nuclease editing comprising:transforming cells with one or more vectors comprising a promoterdriving expression of a nuclease, an inducible promoter drivingtranscription of a guide nucleic acid, and a DNA donor sequence;diluting the transformed cells to a cell concentration to substantiallysingulate the transformed cells on a first substrate; growing the cellsto form colonies on the first substrate; selecting cells from thesubstantially singulated colonies from the first substrate and arrayingthe selected cells on a second substrate; making a replica of the secondsubstrate forming a third substrate; growing and inducing cells on thesecond substrate; growing and inducing cells on the third substrateunder conditions that do not allow genome repair; comparing cell growthon the second and third substrates; and selecting cells that grow on thesecond substrate but do not grow on the third substrate.

In some aspects of the method embodiments, the promoter drivingtranscription the guide nucleic acid is a pL promoter, and in someaspects, the promoter driving expression of the nuclease is an induciblepromoter. In some aspects, the inducible promoter driving expression ofeach of the guide nucleic acid and the nuclease is the same induciblepromoter, and it is a pL promoter. In some aspects, the DNA donorsequence further comprises a PAM-altering sequence, and the methodfurther comprises adding selective agents to medium of the firstsubstrate to select for the one or more vectors. In some aspects, theguide nucleic acid is a guide RNA, and in some aspects the cells arebacteria cells and the engine vector further comprises a recombineeringsystem.

Yet another embodiment of a method provides a method for enrichingedited cells during nucleic acid-guided nuclease editing comprising:transforming cells with one or more vectors comprising a promoterdriving expression of a nuclease, an inducible promoter drivingtranscription of a guide nucleic acid, and a DNA donor sequence;diluting the transformed cells to a cell concentration to substantiallysingulate the transformed cells on a substrate; growing the cells on thesubstrate for between 2 and 200 doublings; initiating editing byinducing the inducible promoter and growing the cells to form colonies;and selecting small colonies from the substantially singulated coloniesfrom the substrate, wherein the induced substantially singulatedcolonies are enriched for edited cells.

And yet an additional embodiment provides a method for enriching editedcells during nucleic acid-guided nuclease editing comprising:transforming cells with one or more vectors comprising a promoterdriving expression of a nuclease, an inducible promoter drivingtranscription of a guide nucleic acid, and a DNA donor sequence;diluting the transformed cells to a cell concentration to substantiallysingulate the transformed cells on a first substrate; growing the cellson the first substrate for between 2 and 200 doublings; initiatingediting by inducing the inducible promoter; and growing the cells toform colonies of terminal size. Some aspects of this embodiment furthercomprise pooling the terminal-size colonies, and some aspects of thisembodiment further comprise picking the terminal-size colonies.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a simplified flow chart of exemplary methods for enrichingand selecting edited cells. FIG. 1B is a plot of optical density vs.time showing the growth curves for edited cells (dotted line) andunedited cells (solid line). FIG. 1C depicts an exemplary inducibleexpression system for regulating gRNA and/or nuclease transcription.

FIG. 2A depicts a prior art, standard protocol for performing nucleicacid-guided nuclease genome editing. FIGS. 2B-2F depict improvedprotocols employing singulation or substantial singulation, induction,and either normalization or cherry picking (e.g., selection) foridentifying edited cells in a population of cells that have undergonenucleic acid-guided nuclease genome editing. FIG. 2B depicts a protocolfor functional deconvolution of the editing process, either by arrayingcells in 96-well plates containing different media or by arraying cellson a culture dish containing different media. FIG. 2C depicts a protocolfor picking colonies from a culture dish, arraying the colonies on a96-well plate, then performing functional deconvolution. FIG. 2E depictsa protocol for cherry picking, and FIG. 2F depicts a protocol used forconfirming that cherry-picking is extremely effective for selecting foredited cells.

FIG. 3A depicts a simplified graphic of a workflow for singulating,editing and normalizing cells in a solid wall device. 3B depicts asimplified graphic of a workflow variation for substantiallysingulating, editing and normalizing cells in a solid wall device. FIG.3C is a photograph of one embodiment of a solid wall device. FIGS. 3D-3Fare photographs of E. coli cells largely singulated (via substantialPoisson distribution) and grown into colonies in microwells in a solidwall device with a permeable bottom at low, medium, and highmagnification, respectively.

FIGS. 4A-4E are photographs of the perforated member and the microwellstherein. FIG. 4F-4R depict the components of three exemplary embodimentsof a singulation assembly comprising retentate and permeate members, aswell as a perforated member/filter/gasket assembly. FIG. 4S-4Z depict anassembled singulation, growth, induction of editing and eithernormalization or cherry-picking module (e.g., “solid wallisolation/induction/normalization module” or “SWIIN”). FIGS. 4AA and 4BBdepict compound SWIIN modules comprising two (FIG. 4AA) or four (FIG.4BB) singulation assemblies. FIG. 4CC is an exemplary pneumaticarchitecture diagram for the SWIIN module described in relation to FIGS.4A-4Z. Finally, FIG. 4DD is an exemplary pneumatic architecture diagramfor a double layer SWIIN.

FIGS. 5A-5D depict a stand-alone, integrated, automated multi-moduleinstrument and components thereof, including a singulation module, withwhich to generate and identify edited cells.

FIG. 6A depicts one embodiment of a rotating growth vial for use withthe cell growth module described herein. FIG. 6B illustrates aperspective view of one embodiment of a rotating growth device in a cellgrowth module housing. FIG. 6C depicts a cut-away view of the cellgrowth module from FIG. 6B. FIG. 6D illustrates the cell growth moduleof FIG. 6B coupled to LED, detector, and temperature regulatingcomponents.

FIG. 7A is a model of the process of tangential flow filtration that isused in the TFF module presented herein. FIG. 7B depicts a top view of alower member of one embodiment of an exemplary TFF device/module. FIG.7C depicts a top-down view of upper and lower members and a membrane ofan exemplary TFF module. FIG. 7D depicts a bottom-up view of upper andlower members and a membrane of an exemplary TFF module. FIGS. 7E-7Hdepict various views of an embodiment of a TFF module havingfluidically-coupled reservoirs for retentate, filtrate, and exchangebuffer.

FIGS. 8A and 8B are top perspective and bottom perspective views,respectively, of flow-through electroporation devices (here, there aresix such devices co-joined). FIG. 8C is a top view of one embodiment ofan exemplary flow-through electroporation device. FIG. 8D depicts a topview of a cross section of the electroporation device of FIG. 8C. FIG.8E is a side view cross section of a lower portion of theelectroporation devices of FIGS. 8C and 8D.

FIG. 9 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising asingulation or substantial singulation/growth/editing and normalizationor cherry picking module (“solid wall isolation/induction/normalizationmodule” or “SWIIN”).

FIG. 10 is a simplified block diagram of an alternative embodiment of anexemplary automated multi-module cell processing instrument comprising asingulation or substantial singulation/growth/editing and normalizationor cherry picking module (“solid wall isolation/induction/normalizationmodule” or “SWIIN”).

FIG. 11A is a map of an exemplary engine vector that may be used in themethods described herein; and FIG. 11B is a map of an exemplary editingvector (with an editing cassette) that may be used in the methodsdescribed herein.

FIG. 12 is a bar graph showing the transformation efficiencies observedfor galK gRNA targeting cassettes under a variety of promoters.

FIG. 13 is a plot of cell growth vs. time, demonstrating that thermalinduction of editing does not impact cell growth or viability.

FIG. 14 depicts results demonstrating repressed gRNA cassettes yieldhigh cell viability/transformation efficiency for three exemplarynucleases.

FIG. 15 illustrates heat maps and growth curves showing the OD at 6hours for uninduced and induced cell populations.

FIG. 16 shows the results of cell colony normalization for E. coli cellsunder various conditions.

FIG. 17A is a photograph of a solid wall device with a permeable bottomon agar, on which yeast cells have been substantially singulated andgrown into clonal colonies.

FIG. 17B presents photographs of yeast colony growth at various timepoints. FIG. 18 is a graph comparing the percentage of editing obtainedfor a standard plating protocol (SPP), and replicates samples using twodifferent conditions in a singulation in a solid wall isolation,induction, and normalization device (SWIIN): the first withLB+arabinose; and the second with SOB followed by SOB+arabinose.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentare intended to be applicable to the additional embodiments describedherein except where expressly stated or where the feature or function isincompatible with the additional embodiments. For example, where a givenfeature or function is expressly described in connection with oneembodiment but not expressly mentioned in connection with an alternativeembodiment, it should be understood that the feature or function may bedeployed, utilized, or implemented in connection with the alternativeembodiment unless the feature or function is incompatible with thealternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and sequencing technology,which are within the skill of those who practice in the art. Suchconventional techniques include polymer array synthesis, hybridizationand ligation of polynucleotides, and detection of hybridization using alabel. Specific illustrations of suitable techniques can be had byreference to the examples herein. However, other equivalent conventionalprocedures can, of course, also be used. Such conventional techniquesand descriptions can be found in standard laboratory manuals such asGreen, et al., eds., Genome Analysis: A Laboratory Manual Series (Vols.I-IV) (1999); Weiner, Gabriel, Stephens, eds., Genetic Variation: ALaboratory Manual (2007); Dieffenbach, Dveksler, eds., PCR Primer: ALaboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: AMolecular Cloning Manual (2003); Mount, Bioinformatics: Sequence andGenome Analysis (2004); Sambrook and Russell, Condensed Protocols fromMolecular Cloning: A Laboratory Manual (2006); Stryer, Biochemistry (4thEd.) W.H. Freeman, New York N.Y. (1995); Gait, “OligonucleotideSynthesis: A Practical Approach” (1984), IRL Press, London; Nelson andCox, Lehninger, Principles of Biochemistry 3^(rd) Ed., W. H. FreemanPub., New York, N.Y. (2000); Berg et al., Biochemistry, 5^(th) Ed., W.H.Freeman Pub., New York, N.Y. (2002); Doyle & Griffiths, eds., Cell andTissue Culture: Laboratory Procedures in Biotechnology, Doyle &Griffiths, eds., John Wiley & Sons (1998); G. Hadlaczky, ed. MammalianChromosome Engineering—Methods and Protocols, Humana Press (2011); andLanza and Klimanskaya, eds., Essential Stem Cell Methods, Academic Press(2011), all of which are herein incorporated in their entirety byreference for all purposes. CRISPR-specific techniques can be found in,e.g., Appasani and Church, Genome Editing and Engineering From TALENsand CRISPRs to Molecular Surgery (2018); and Lindgren and Charpentier,CRISPR: Methods and Protocols (2015); both of which are hereinincorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”and/or “outer” that may be used herein merely describe points ofreference and do not necessarily limit embodiments of the presentdisclosure to any particular orientation or configuration. Furthermore,terms such as “first,” “second,” “third,” etc., merely identify one of anumber of portions, components, steps, operations, functions, and/orpoints of reference as disclosed herein, and likewise do not necessarilylimit embodiments of the present disclosure to any particularconfiguration or orientation.

Additionally, the terms “approximately,” “proximate,” “minor,” andsimilar terms generally refer to ranges that include the identifiedvalue within a margin of 20%, 10% or preferably 5% in certainembodiments, and any values therebetween.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, methods and cell populations that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of ordinary skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotides thatare hydrogen bonded to one another with thymine or uracil residueslinked to adenine residues by two hydrogen bonds and cytosine andguanine residues linked by three hydrogen bonds. In general, a nucleicacid includes a nucleotide sequence described as having a “percentcomplementarity” or “percent homology” to a specified second nucleotidesequence. For example, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TTAGCTGG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

As used herein the term “donor DNA” or “donor nucleic acid” refers tonucleic acid that is designed to introduce a DNA sequence modification(insertion, deletion, substitution) into a locus by homologousrecombination using nucleic acid-guided nucleases. For homology-directedrepair, the donor DNA must have sufficient homology to the regionsflanking the “cut site” or site to be edited in the genomic targetsequence. The length of the homology arm(s) will depend on, e.g., thetype and size of the modification being made. For example, the donor DNAwill have at least one region of sequence homology (e.g., one homologyarm) to the genomic target locus. In many instances and preferably, thedonor DNA will have two regions of sequence homology (e.g., two homologyarms) to the genomic target locus. Preferably, an “insert” region or“DNA sequence modification” region—the nucleic acid modification thatone desires to be introduced into a genome target locus in a cell-willbe located between two regions of homology. The DNA sequencemodification may change one or more bases of the target genomic DNAsequence at one specific site or multiple specific sites. A change mayinclude changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,150, 200, 300, 400, or 500 or more base pairs of the target sequence. Adeletion or insertion may be a deletion or insertion of 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 ormore base pairs of the target sequence. The donor DNA optionally furtherincludes an alteration to the target sequence, e.g., a PAM mutation,that prevents binding of the nuclease at the PAM or spacer in the targetsequence after editing has taken place.

As used herein, “enrichment” refers to enriching for edited cells bysingulation or substantial singulation of cells, initial growth of cellsinto cell colonies, inducing editing, and growing the cell colonies intoterminal-sized colonies (e.g., saturation or normalization of colonygrowth). As used herein, “cherry picking” or “selection of edited cells”refers to the process of using a combination of singulation orsubstantial singulation, initial growth of cells into colonies,induction of editing, then using cell growth-measured by colony size,concentration of metabolites or waste products, or other characteristicsthat correlate with the rate of growth of the cells—to select for cellsthat have been edited based on editing-induced growth delay.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive, or inducible. In the methods describedherein the promoters driving transcription of the gRNAs is inducible.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampilcillin/carbenicillin,kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin,bleomycin, streptomycin, puromycin, hygromycin, blasticidin, and G418may be employed. In other embodiments, selectable markers include, butare not limited to human nerve growth factor receptor (detected with aMAb, such as described in U.S. Pat. No. 6,365,373); truncated humangrowth factor receptor (detected with MAb); mutant human dihydrofolatereductase (DHFR; fluorescent MTX substrate available); secreted alkalinephosphatase (SEAP; fluorescent substrate available); human thymidylatesynthase (TS; confers resistance to anti-cancer agentfluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1;conjugates glutathione to the stem cell selective alkylator busulfan;chemoprotective selectable marker in CD34+ cells); CD24 cell surfaceantigen in hematopoietic stem cells; rhamnose; human CAD gene to conferresistance to N-phosphonacetyl-L-aspartate (PALA); human multi-drugresistance-1 (MDR-1; P-glycoprotein surface protein selectable byincreased drug resistance or enriched by FACS); human CD25 (IL-2α;detectable by MAb-FITC); Methylguanine-DNA methyltransferase (MGMT;selectable by carmustine); and Cytidine deaminase (CD; selectable byAra-C). “Selective medium” as used herein refers to cell growth mediumto which has been added a chemical compound or biological moiety thatselects for or against selectable markers.

As used herein, the terms “singulation” or “singulate” mean to separateindividual cells so that each cell (and the colonies formed from eachcell) will be separate from other cells; for example, a single cell in asingle microwell, or 100 single cells each in its own microwell.“Singulation” or “singulated cells” result in one embodiment, from aPoisson distribution in arraying cells. The terms “substantiallysingulated”, “largely singulated”, and “substantial singulation” meancells are largely separated from one another, in small groups orbatches. That is, when 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or up to50—but preferably 10 or less cells—are delivered to a microwell.“Substantially singulated” or “largely singulated” result, in oneembodiment, from a “substantial Poisson distribution” in arraying cells.With more complex libraries of edits—or with libraries that may compriselethal edits or edits with greatly-varying fitness effects—it ispreferred that cells be singulated via a Poisson distribution.

The terms “target genomic DNA sequence”, “target sequence”, or “genomictarget locus” refer to any locus in vitro or in vivo, or in a nucleicacid (e.g., genome) of a cell or population of cells, in which a changeof at least one nucleotide is desired using a nucleic acid-guidednuclease editing system. The target sequence can be a genomic locus orextrachromosomal locus.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, YACs, BACs, mammalian synthetic chromosomes,and the like. As used herein, the phrase “engine vector” comprises acoding sequence for a nuclease—optionally under the control of aninducible promoter—to be used in the nucleic acid-guided nucleasesystems and methods of the present disclosure. The engine vector mayalso comprise, in a bacterial system, the λ Red recombineering system oran equivalent thereto, as well as a selectable marker. As used hereinthe phrase “editing vector” comprises a donor nucleic acid, including analteration to the target sequence which prevents nuclease binding at aPAM or spacer in the target sequence after editing has taken place, anda coding sequence for a gRNA under the control of an inducible promoter.The editing vector may also comprise a selectable marker and/or abarcode. In some embodiments, the engine vector and editing vector maybe combined; that is, the contents of the engine vector may be found onthe editing vector.

Inducible Editing in Nucleic Acid-Guided Nuclease Genome SystemsGenerally

The present disclosure provides instruments, modules and methods fornucleic acid-guided nuclease genome editing that provide 1) enhancedobserved editing efficiency of nucleic acid-guided nuclease editingmethods, and 2) improvement in screening for and detecting cells whosegenomes have been properly edited, including high-throughput screeningtechniques. In current protocols employing nuclease systems,constitutively-expressed nuclease components typically are used to drivehigh-efficiency editing. However, in pooled or multiplex formats,constitutive expression of editing components can lead to selectiveenrichment of cells that are not edited due to the lack of double-strandDNA breaks that occur during editing. Moreover, constitutively-expressednucleic acid-guided nuclease components (e.g., the nuclease and guidenucleic acid) expose the freshly-transformed, physiologically fragilecells to editing immediately, resulting in compromised viability.Presented herein are tools for tightly-regulated expression of nucleicacid-guided nuclease editing system components including tightregulation of transcription of the guide nucleic acid and, optionallyand preferably, the nuclease (or the nuclease and, optionally andpreferably, the guide nucleic acid) allowing for separation of theprocesses of transformation and editing. Additionally, the methodsdescribed herein take advantage of singulation (separating cells andgrowing them into clonal colonies) and either normalization of cellcolonies or cherry picking of slow-growing colonies. Singulation orsubstantial singulation, initial growth, followed by induction ofediting and normalization overcomes growth bias from unedited cells, andsubstituting cherry picking for normalization allows for directselection of edited cells. The instruments, modules, and methods may beapplied to all cell types including, archaeal, prokaryotic, andeukaryotic (e.g., yeast, fungal, plant and animal) cells.

The instruments, modules, and methods described herein employ editingcassettes comprising a guide RNA (gRNA) sequence covalently linked to adonor DNA sequence where the gRNA is under the control of an induciblepromoter (e.g., the editing cassettes are CREATE cassettes; see U.S.patent application Ser. No. 9/982,278, issued 29 May 2019 and Ser. No.10/240,167, issued 26 Mar. 2019; Ser. No. 10/266,849, issued 23 Apr.2019; and U.S. Ser. No. 15/948,785, filed 9 Apr. 2018; Ser. No.16/275,439, filed 14 Feb. 2019; and Ser. No. 16/275,465, filed 14 Feb.2019, all of which are incorporated by reference in their entirety), Theediting cassettes provide increased transformation efficiency andcontrol over the timing and duration of the editing process. Thedisclosed methods allow for cells to be transformed, substantiallysingulated, grown for several doublings, after which editing in thecells is induced. The combination of process effectively negates theeffect of unedited cells taking over the cell population. Thecombination of substantially singulating cells, then allowing forinitial growth followed by inducing transcription of the gRNA (and/ornuclease) and either normalization of cell colonies or cherry pickingcells leads to 2-250×, 10-225×, 25-200×, 40-175×, 50-150×, 60-100×, or50-100× gains in identifying edited cells over prior art methods andallows for generation of arrayed or pooled edited cells comprising celllibraries with edited genomes. Additionally, the methods may beleveraged to create iterative editing systems to generate combinatoriallibraries of cells with two to many edits in each cellular genome. Theinducible gRNA constructs (and/or inducible nuclease constructs) andmethods for using “pulsed” exposure of the cells to active editingcomponents 1) allow for the cells to be arrayed (e.g., largelysingulated) prior to initiation of the editing procedure, 2) decreaseoff-target activity, 3) allow for identification of rare cell edits, and4) enrich for edited cells or permit high-throughput screeningapplications to identify editing activity using cell growth as a proxyfor editing, by, e.g., measuring optical density, colony size, ormetabolic by-products or other characteristics thereby enriching theedited cell population.

The instruments, compositions and methods described herein improveCRISPR editing systems in which nucleic acid-guided nucleases (e.g.,RNA-guided nucleases) are used to edit specific target regions in anorganism's genome. A nucleic acid-guided nuclease complexed with anappropriate synthetic guide nucleic acid in a cell can cut the genome ofthe cell at a desired location. The guide nucleic acid helps the nucleicacid-guided nuclease recognize and cut the DNA at a specific targetsequence. By manipulating the nucleotide sequence of the guide nucleicacid, the nucleic acid-guided nuclease may be programmed to target anyDNA sequence for cleavage as long as an appropriate protospacer adjacentmotif (PAM) is nearby. In certain aspects, the nucleic acid-guidednuclease editing system may use two separate guide nucleic acidmolecules that combine to function as a guide nucleic acid, e.g., aCRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In otheraspects, the guide nucleic acid may be a single guide nucleic acid thatincludes both the crRNA and tracrRNA sequences or a single guide nucleicacid that does not require a tracrRNA. In the compositions and methodsdescribed herein, if two separate RNA molecules are combined to functionas a guide nucleic acid, at least one of the guide nucleic acidcomponents is under the control of an inducible promoter. If a singleguide nucleic acid is employed, the single guide nucleic acid is underthe control of an inducible promoter.

In general, a guide nucleic acid (e.g., gRNA) complexes with acompatible nucleic acid-guided nuclease and can then hybridize with atarget sequence, thereby directing the nuclease to the target sequence.A guide nucleic acid can be DNA or RNA; alternatively, a guide nucleicacid may comprise both DNA and RNA. In some embodiments, a guide nucleicacid may comprise modified or non-naturally occurring nucleotides. Incases where the guide nucleic acid comprises RNA, the gRNA is encoded bya DNA sequence on a polynucleotide molecule such as a plasmid, linearconstruct, or resides within an editing cassette and is preferably underthe control of an inducible promoter.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence (the portion of theguide nucleic acid that hybridizes with the target sequence) is about ormore than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides inlength. In some embodiments, a guide sequence is less than about 75, 50,45, 40, 35, 30, 25, 20 nucleotides in length. Preferably the guidesequence is 10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or20 nucleotides in length.

In the present methods and compositions, the guide nucleic acid isprovided as a sequence to be expressed from a plasmid or vector andcomprises both the guide sequence and the scaffold sequence as a singletranscript under the control of an inducible promoter. Alternatively,the guide nucleic acids may be transcribed from two separate sequences,at least one of which is under the control of an inducible promoter. Theguide nucleic acid can be engineered to target a desired target DNAsequence by altering the guide sequence so that the guide sequence iscomplementary to the target DNA sequence, thereby allowing hybridizationbetween the guide sequence and the target DNA sequence. In general, togenerate an edit in the target DNA sequence, the gRNA/nuclease complexbinds to a target sequence as determined by the guide RNA, and thenuclease recognizes a protospacer adjacent motif (PAM) sequence adjacentto the target sequence. The target sequence can be any polynucleotide(either DNA or RNA) endogenous or exogenous to a prokaryotic oreukaryotic cell, or in vitro. For example, the target sequence can be apolynucleotide residing in the nucleus of a eukaryotic cell. A targetsequence can be a sequence encoding a gene product (e.g., a protein)and/or a non-coding sequence (e.g., a regulatory polynucleotide, anintron, a PAM, or “junk” DNA).

The guide nucleic acid may be part of an editing cassette that encodesthe donor nucleic acid; that is, the editing cassette may be a CREATEcassette (see, e.g U.S. patent Ser. No. 9/982,278, issued 29 May 2019and Ser. No. 10/240,167, issued 26 Mar. 2019; Ser. No. 10/266,849,issued 23 Apr. 2019; and U.S. Ser. No. 15/948,785, filed 9 Apr. 2018;Ser. No. 16/275,439, filed 14 Feb. 2019; and Ser. No. 16/275,465, filed14 Feb. 2019 all of which are incorporated by reference in theirentirety). The guide nucleic acid and the donor nucleic acid may be andtypically are under the control of a single (in this case, preferablyinducible) promoter. Again, note that either the guide nucleic acid orthe nuclease must be under the control of an inducible promoter, and insome embodiments and preferably, both the guide nucleic acid andnuclease are under the control of an inducible promoter. Indeed, it isimportant that the inducible promoter(s) controlling transcription ofthe guide nucleic acid and/or—most preferably, and—the nuclease aretightly controlled. In particular, it is important that the nucleaseactivity be controlled to avoid significant bias and loss in efficiencyprior to singulation and singulated outgrowth. Thus, the extent to whichthe editing machinery is able to achieve “OFF” status is importantindependent of the singulation or substantial singulation process.Alternatively, the guide nucleic acid may not be part of the editingcassette and instead may be encoded on the engine or editing vectorbackbone. For example, a sequence coding for a guide nucleic acid can beassembled or inserted into a vector backbone first, followed byinsertion of the donor nucleic acid. In other cases, the donor nucleicacid can be inserted or assembled into a vector backbone first, followedby insertion of the sequence coding for the guide nucleic acid. In yetother cases, the sequence encoding the guide nucleic acid and the donornucleic acid (inserted, for example, in an editing cassette) aresimultaneously but separately inserted or assembled into a vector. Inyet other embodiments and preferably, the sequence encoding the guidenucleic acid and the sequence encoding the donor nucleic acid are bothincluded in the editing cassette.

The target sequence is associated with a PAM, which is a shortnucleotide sequence recognized by the gRNA/nuclease complex. The precisePAM sequence and length requirements for different nucleic acid-guidednucleases vary; however, PAMs typically are 2-7 base-pair sequencesadjacent or in proximity to the target sequence and, depending on thenuclease, can be 5′ or 3′ to the target sequence. Engineering of thePAM-interacting domain of a nucleic acid-guided nuclease may allow foralteration of PAM specificity, improve target site recognition fidelity,decrease target site recognition fidelity, and increase the versatilityof a nucleic acid-guided nuclease. In certain embodiments, the genomeediting of a target sequence both introduces a desired DNA change to atarget sequence, e.g., the genomic DNA of a cell, and removes, mutates,or renders inactive a proto-spacer (PAM) region in the target sequence;that is, the donor DNA often includes an alteration to the targetsequence that prevents binding of the nuclease at the PAM in the targetsequence after editing has taken place. Rendering the PAM at the targetsequence inactive precludes additional editing of the cell genome atthat target sequence, e.g., upon subsequent exposure to a nucleicacid-guided nuclease complexed with a synthetic guide nucleic acid inlater rounds of editing. Thus, cells having the desired target sequenceedit and an altered PAM can be selected using a nucleic acid-guidednuclease complexed with a synthetic guide nucleic acid complementary tothe target sequence. Cells that did not undergo the first editing eventwill be cut rendering a double-stranded DNA break, and thus will notcontinue to be viable. The cells containing the desired target sequenceedit and PAM alteration will not be cut, as these edited cells no longercontain the necessary PAM site and will continue to grow and propagate.

The range of target sequences that nucleic acid-guided nucleases canrecognize is constrained by the need for a specific PAM to be locatednear the desired target sequence. As a result, it often can be difficultto target edits with the precision that is necessary for genome editing.It has been found that nucleases can recognize some PAMs very well(e.g., canonical PAMs), and other PAMs less well or poorly (e.g.,non-canonical PAMs). Because the methods disclosed herein allow foridentification of edited cells in a large background of unedited cells,the methods allow for identification of edited cells where the PAM isless than optimal; that is, the methods for identifying edited cellsherein allow for identification of edited cells even if editingefficiency is very low. Additionally, the present methods expand thescope of target sequences that may be edited since edits are morereadily identified, including cells where the genome edits areassociated with less functional PAMs.

As for the nuclease component of the nucleic acid-guided nucleaseediting system, the polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcells, such as archaeal, prokaryotic or eukaryotic cells. Eukaryoticcells can be yeast, fungi, algae, plant, animal, or human cells.Eukaryotic cells may be those of or derived from a particular organism,such as a mammal, including but not limited to human, mouse, rat,rabbit, dog, or non-human mammal including non-human primate. The choiceof nucleic acid-guided nuclease to be employed depends on many factors,such as what type of edit is to be made in the target sequence andwhether an appropriate PAM is located close to the desired targetsequence. Nucleases of use in the methods described herein include butare not limited to Cas 9, Cas 12/CpfI, MAD2, or MAD7 or other MADzymes.As with the guide nucleic acid, the nuclease may be encoded by a DNAsequence on a vector (e.g., the engine vector) and be under the controlof a constitutive or, preferably, an inducible promoter. Again, at leastone of and preferably both of the nuclease and guide nucleic acid areunder the control of an inducible promoter. In some embodiments, thesequence encoding the nuclease is under the control of an induciblepromoter, and the inducible promoter may be separate from but the sameas the inducible promoter controlling transcription of the guide nucleicacid; that is, a separate inducible promoter drives the transcription ofthe nuclease and guide nucleic acid sequences but the two induciblepromoters may be the same type of inducible promoter (e.g., both are pLpromoters). Alternatively, the inducible promoter controlling expressionof the nuclease may be different from the inducible promoter controllingtranscription of the guide nucleic acid; that is, e.g., the nuclease maybe under the control of the pBAD inducible promoter, and the guidenucleic acid may be under the control of the pL inducible promoter.Again, note that it is important that the inducible promoter(s)controlling transcription of the guide nucleic acid and the nuclease aretightly controlled. In particular, it is important that the nucleaseactivity be controlled to avoid significant bias and loss in efficiencyprior to singulation and the singulated outgrowth process. Thus, theextent to which the editing machinery is able to achieve “OFF” status isimportant independent of the singulation or substantial singulationprocess.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid. In some embodiments, the donor nucleic acid is onthe same polynucleotide (e.g., vector or editing (CREATE) cassette) asthe guide nucleic acid. The donor nucleic acid is designed to serve as atemplate for homologous recombination with a target sequence nicked orcleaved by the nucleic acid-guided nuclease as a part of thegRNA/nuclease complex. A donor nucleic acid polynucleotide may be of anysuitable length, such as about or more than about 30, 35, 40, 45, 50,75, 100, 150, 200, 500, 1000, 2500, 5000 nucleotides or more in length.In certain preferred aspects, the donor nucleic acid can be provided asan oligonucleotide of between 40-300 nucleotides, more preferablybetween 50-250 nucleotides. The donor nucleic acid comprises a regionthat is complementary to a portion of the target sequence (e.g., ahomology arm). When optimally aligned, the donor nucleic acid overlapswith (is complementary to) the target sequence by, e.g., about 10, 15,20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides. In manyembodiments, the donor nucleic acid comprises two homology arms (regionscomplementary to the target sequence) flanking the mutation ordifference between the donor nucleic acid and the target template. Thedonor nucleic acid comprises at least one mutation or alterationcompared to the target sequence, such as an insertion, deletion,modification, or any combination thereof compared to the targetsequence.

Often the donor nucleic acid is provided as an editing cassette, whichis inserted into a vector backbone where the vector backbone maycomprise a promoter driving transcription of the gRNA and the codingsequence of the gRNA, or the vector backbone may comprise a promoterdriving the transcription of the gRNA but not the gRNA itself. Moreover,there may be more than one, e.g., two, three, four, or more guidenucleic acid/donor nucleic acid cassettes inserted into an enginevector, where the guide nucleic acids are under the control of separate,different promoters, separate, like promoters, or where all guidenucleic acid/donor nucleic acid pairs are under the control of a singlepromoter. (See, e.g., U.S. Ser. No. 16/275,465, filed 14 Feb. 2019,drawn to multiple CREATE cassettes.) The promoter driving transcriptionof the gRNA and the donor nucleic acid (or driving more than onegRNA/donor nucleic acid pair) is preferably an inducible promoter andthe promoter driving transcription of the nuclease is optionally aninducible promoter as well. That is, at least one of—and preferablyboth—the guide nucleic acid (gRNA) and the nuclease is under control ofan inducible promoter.

Inducible editing is advantageous in that substantially or largelysingulated cells can be grown for several to many cell doublings beforeediting is initiated, which increases the likelihood that cells withedits will survive, as the double-strand cuts caused by active editingare largely toxic to the cells. This toxicity results both in cell deathin the edited colonies, as well as a lag in growth for the edited cellsthat do survive but must repair and recover following editing. However,once the edited cells have a chance to recover, the size of the coloniesof the edited cells will eventually catch up to the size of the coloniesof unedited cells (e.g., the process of “normalization” or growingcolonies to “terminal size”; see, e.g., FIG. 1B described infra).

In addition to the donor nucleic acid, an editing cassette may compriseone or more primer sites. The primer sites can be used to amplify theediting cassette by using oligonucleotide primers; for example, if theprimer sites flank one or more of the other components of the editingcassette.

Also, as described above, the donor nucleic acid may comprise—inaddition to the at least one mutation relative to a target sequence—oneor more PAM sequence alterations that mutate, delete or render inactivethe PAM site in the target sequence. The PAM sequence alteration in thetarget sequence renders the PAM site “immune” to the nucleic acid-guidednuclease and protects the target sequence from further editing insubsequent rounds of editing if the same nuclease is used.

The editing cassette also may comprise a barcode. A barcode is a uniqueDNA sequence that corresponds to the donor DNA sequence such that thebarcode can identify the edit made to the corresponding target sequence.The barcode can comprise greater than four nucleotides. In someembodiments, the editing cassettes comprise a collection of donornucleic acids representing, e.g., gene-wide or genome-wide libraries ofdonor nucleic acids. The library of editing cassettes is cloned intovector backbones where, e.g., each different donor nucleic acid designis associated with a different barcode, or, alternatively, eachdifferent cassette molecule is associate with a different barcode.

Additionally, in some embodiments, an expression vector or cassetteencoding components of the nucleic acid-guided nuclease system furtherencodes a nucleic acid-guided nuclease comprising one or more nuclearlocalization sequences (NLSs), such as about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the engineerednuclease comprises NLSs at or near the amino-terminus, NLSs at or nearthe carboxy-terminus, or a combination.

Exemplary Workflows for Editing, Enrichment, and Selection of EditedCells

The methods described herein provide enhanced observed editingefficiency of nucleic acid-guided nuclease editing methods as the resultof a combination of singulation or substantial singulation, initial cellgrowth, induction of editing, and either normalization of the resultingcell colonies or cherry picking slow-growing cell colonies. Thecombination of the singulation or substantial singulation, initial cellgrowth, induction of editing and normalization processes overcomes thegrowth bias in favor of unedited cells—and the fitness effects ofediting (including differential editing rates)—thus allowing all cells“equal billing” with one another. The combination of singulation orsubstantial singulation, initial cell growth, induction of editing, andcherry picking allows for direct selection of edited colonies of cells.The result of the methods described herein is that even in nucleicacid-guided nuclease systems where editing is not optimal—such as insystems where non-canonical PAMs are targeted—there is an increase inthe observed editing efficiency; that is, edited cells can be identifiedeven in a large background of unedited cells. Observed editingefficiency can be improved up to 80% or more. Singulating, initialgrowth, induction of editing, and normalization of cell colonies orcherry picking of cell colonies leads to 2-250×, 10-225×, 25-200×,40-175×, 50-150×, 60-100×, or 5-100× gains in identifying edited cellsover prior art methods and allows for the generation of arrayed orpooled edited cells comprising genome libraries. Additionally, theinstruments, modules and methods may be leveraged to create iterativeediting systems to generate combinatorial libraries, identify rare celledits, and enable high-throughput enrichment applications to identifyediting activity.

FIG. 1A shows simplified flow charts for two alternative exemplarymethods 100 a and 100 b for singulating cells for enrichment (100 a) andfor cherry picking (100 b). Looking at FIG. 1A, method 100 begins bytransforming cells 110 with the components necessary to perform nucleicacid-guided nuclease editing. For example, the cells may be transformedsimultaneously with separate engine and editing vectors; the cells mayalready be transformed with an engine vector expressing the nuclease(e.g., the cells may have already been transformed with an engine vectoror the coding sequence for the nuclease may be stably integrated intothe cellular genome) such that only the editing vector needs to betransformed into the cells; or the cells may be transformed with asingle vector comprising all components required to perform nucleicacid-guided nuclease genome editing.

A variety of delivery systems can be used to introduce (e.g., transformor transfect) nucleic acid-guided nuclease editing system componentsinto a host cell 110. These delivery systems include the use of yeastsystems, lipofection systems, microinjection systems, biolistic systems,virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acidconjugates, virions, artificial virions, viral vectors, electroporation,cell permeable peptides, nanoparticles, nanowires, exosomes.Alternatively, molecular trojan horse liposomes may be used to delivernucleic acid-guided nuclease components across the blood brain barrier.Of interest, particularly in the context of an automated multi-modulecell editing instrument is the use of electroporation, particularlyflow-through electroporation (either as a stand-alone instrument or as amodule in an automated multi-module system) as described in, e.g., U.S.Ser. No. 16/147,120, filed 28 Sep. 2018; Ser. No. 16/147,353, filed 28Sep. 2018; Ser. No. 16/147,865, filed 30 Sep. 2018; and Ser. No.16/147,871, filed 30 Sep. 2018. If the solid wall singulation orsubstantial singulation/growth/induction of editing and normalizationmodule is one module in an automated multi-module cell editinginstrument as described herein infra, the cells are likely transformedin an automated cell transformation module.

After the cells are transformed with the components necessary to performnucleic acid-guided nuclease editing, the cells are substantially orlargely singulated 120; that is, the cells are diluted (if necessary) ina liquid culture medium so that the cells, when delivered to a substratefor singulation, are substantially separated from one another and canform colonies that are substantially separated from one another. Forexample, if a solid wall device is used (described infra in relation toFIGS. 3A-3E and 4A-4CC), the cells are diluted such that when deliveredto the solid wall device the cells fill the microwells of the solid walldevice in a Poisson or substantial Poisson distribution. In one example(illustrated in FIG. 3A), singulation is accomplished when an average of½ cell is delivered to each microwell; that is, where some microwellscontain one cell and other microwells contain no cells. Alternatively, asubstantial Poisson distribution of cells occurs when two to several (2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or up to 50, but preferably 10 or less,or preferably 5 or less) cells are delivered to a microwell (illustratedin FIG. 3B).

Once the cells have been substantially or largely singulated 120, thecells are allowed to grow to, e.g., between 2 and 130, or between 5 and120, or between 10 and 100 doublings, establishing clonal colonies 140.After colonies are established, editing is induced 130, e.g., where oneor more inducible promoters driving transcription of one or both of thegRNA and the nuclease (preferably both), as well as, e.g., the λ redrecombination system components in bacterial systems. For an exemplaryinducible system, see FIG. 1C described in detail infra. Once editing iscomplete, in enrichment method 100 a the cells are grown into coloniesof terminal size 150; that is, the colonies arising from thesubstantially or largely singulated cells are grown into colonies to apoint where cell growth has peaked and is normalized or saturated forboth edited and unedited cells. Normalization occurs as the nutrients inthe medium around a growing cell colony are depleted and/or cell growthfills the microwells and further growth is physically constrained. Theterminal-size colonies are pooled 160 by, e.g., scraping the coloniesoff a plate comprising solid medium or other substrate or by flushingclonal cell colonies from microwells in a solid wall device or module topool the cells from the normalized cell colonies. Again, becausesingulation or substantial singulation overcomes growth bias fromunedited cells or cells exhibiting fitness effects as the result ofedits made, singulation or substantial singulation, initial growth,induction of editing, and normalization alone enriches the totalpopulation of cells with cells that have been edited; that is,singulation or substantial singulation combined with initial cellgrowth, induced editing and normalization (e.g., growing colonies toterminal size) allows for high-throughput enrichment of edited cells.

Method 100 b shown in FIG. 1A is similar to the method 100 a in thatcells of interest are transformed 110 with the components necessary toperform nucleic acid-guided nuclease editing. As described above, thecells may be transformed simultaneously with both the engine and editingvectors, the cells may already be expressing the nuclease (e.g., thecells may have already been transformed with an engine vector or thecoding sequence for the nuclease may be stably integrated into thecellular genome) such that only the editing vector needs to betransformed into the cells, or the cells may be transformed with asingle vector comprising all components required to perform nucleicacid-guided nuclease genome editing. Further, if the singulation orsubstantial singulation of cells, initial cell growth, induced editing,and either normalization or cherry-picking module (“solid wallisolation/induction/normalization module” or “SWIIN”) is one module inan automated multi-module cell editing instrument, cell transformationmay be performed in an automated transformation module as described inrelation to FIGS. 8A-8E below.

After the cells are transformed 110 with the components necessary toperform nucleic acid-guided nuclease editing, the cells are diluted (ifnecessary) in liquid medium so that the cells, when delivered to asingulation device or module, are separated from one another and canform colonies that are separated from one another. For example, if asolid wall device is used (described in relation to FIGS. 3A-3E and4A-4CC) the cells are diluted such that when delivered to the solid walldevice, the cells fill the microwells of the solid wall device in aPoisson or substantial Poisson distribution.

Once the cells have been substantially or largely singulated 120, thecells grown to, e.g., between 2 and 150, or between 5 and 120, orbetween 10 and 100 doublings, establishing clonal colonies 130. Aftercolonies are established, editing is induced 140 by, e.g., activatinginducible promoters that control transcription of at least one of thegRNA and nuclease (as described above, preferably both), and, in thecase of bacteria, a recombineering system. Once editing is induced 140,many of the edited cells in the clonal colonies die due to thedouble-strand DNA breaks that occur during the editing process; however,in a percentage of edited cells, the genome is edited and thedouble-strand break is properly repaired. When allowed to recover, theseedited cells start growing and re-establish cell populations within eachisolated partition or colony; however, the growth of edited coloniestends to lag behind the growth of clonal colonies where an edit has nottaken place (e.g., cell “escapees”). If growth of these colonies ismonitored 170, the small or slow-growing colonies (edited cells) may beidentified and then selected or cherry picked 180 based on theobservable size differentials of the colonies.

FIG. 1B is a plot of OD versus time for unedited cells (solid line)versus edited cells (dashed line). FIG. 1B shows how normalization ofedited and non-edited colonies takes place. Note that the OD (e.g.,growth) of the edited cells lags behind the unedited cells initially,but eventually catches up due, e.g., to unedited cells exhausting thenutrients in the medium, becoming physically constrained within amicrowell or other confined growth area (such as a droplet), orotherwise exiting log-phase growth. The cell colonies are allowed togrow long enough for the growth of the edited colonies to catch up with(approximate the size of, e.g., number of cells in) the uneditedcolonies.

FIG. 1C depicts an exemplary inducible expression system—in thisexample, the pL inducible system—for regulating gRNA activity. At thetop of FIG. 1C there is shown a portion of an exemplary engine vector111 comprising an origin of replication 112, a promoter 114 drivingexpression of the c1857 repressor gene 116, and a first pL promoter 118driving expression of a nuclease 121. At the top of FIG. 1C there isalso seen a portion of an exemplary editing vector 131, comprising anorigin of replication 132, and a second pL promoter 134 drivingtranscription of an editing cassette 136 (e.g., a CREATE cassette) whichincludes a coding sequences for both a gRNA and a donor DNA. The middleillustration of FIG. 1C depicts the product 124 of the c1857 repressorgene 116 on the engine vector 111 actively repressing the first pLpromoter 118 driving transcription of the nuclease 121 and the second pLpromoter 134 driving transcription of the editing cassette 136 on theediting vector 131. Finally, the bottom illustration of FIG. 1C depictsthe protein product 126 of the c1857 repressor gene 116 on the enginevector 111 unfolding/degrading due to increased temperature. Theunfolded or degraded protein product 126 cannot bind first pL promoter118 or second pL promoter 134; thus, pL promoter 118 is active anddrives transcription of the nuclease 121 on engine vector 111 and pLpromoter 134 is active and drives transcription of the editing cassette136 on the editing vector 131.

In FIG. 1C, transcription of both the nuclease and the gRNA are undercontrol of a pL promoter; however, in other embodiments, differentinducible promoters may be used to drive transcription of the nucleaseand gRNA or in some embodiments only one of the nuclease and gRNA areunder control of an inducible promoter. For example, the pL and pBADpromoters are shown in relation to the exemplary engine and editingvectors in FIGS. 11A and 11B, and a number of gene regulation controlsystems have been developed for the controlled expression of genes inplant, microbe and animal cells, including mammalian cells. Thesesystems include the tetracycline-controlled transcriptional activationsystem (Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, Calif.); Bujard andGossen, PNAS, 89(12):5547-5551 (1992)), the Lac Switch Inducible system(Wyborski et al., Environ Mol Mutagen, 28(4):447-58 (1996); DuCoeur etal., Strategies 5(3):70-72 (1992); and U.S. Pat. No. 4,833,080), theecdysone-inducible gene expression system (No et al., PNAS,93(8):3346-3351 (1996)), the cumate gene-switch system (Mullick et al.,BMC Biotechnology, 6:43 (2006)), and the tamoxifen-inducible geneexpression (Zhang et al., Nucleic Acids Research, 24:543-548 (1996)) aswell as others. However, the pL promoter is a particularly usefulinducible promoter because the pL promoter is activated by an increasein temperature, to, e.g., 42° C., and deactivated by returning thetemperature to, e.g., 30° C. With other inducible systems that areactivated by the presence or absence of a particular molecular compound,activating the inducible promoter requires addition of or removal of amolecular compound from the culture medium, thus requiring liquidhandling, medium exchange, wash steps and the like.

FIG. 2A depicts a standard, conventional, prior art protocol 200 forperforming nucleic acid-guided nuclease genome editing whereconstitutively-expressed nuclease components typically are used to drivehigh efficiency editing. In FIG. 2A, a library or collection of editingvectors 202 is introduced 203 (e.g., electroporated) into cultured cells204 that comprise a coding sequence for a nuclease under the control ofa constitutive or inducible promoter. In some embodiments, the codingsequence for the nuclease is contained on an “engine vector”, althoughin other embodiments the coding sequence for the nuclease may beintegrated into the cell genome. In yet another alternative, thecomponents of the engine and editing vectors may be combined. Theediting vectors 202 comprise an editing sequence comprising a sequencefor a desired edit in a nucleic acid sequence endogenous to the cell aswell as an optional PAM-altering sequence (most often a sequence thatdisables the PAM at the target sequence in the genome), a codingsequence for a gRNA under the control of a constitutive promoter, and aselectable marker. Once the cells have been transformed with the editingvectors, the cells are plated 205 on selective medium 206 to select forcells that have both the engine and the editing vectors and the cellsare grown until colonies form. Cells are then picked 207 from thecolonies and grown in, e.g., 96-well plates 208 to be prepared forgenome or plasmid sequencing. The cells that grow on plate 206 with theselective medium should comprise both the engine and editing vectors;however, it is likely that in some cells the nucleic acid-guided editingcomponents may be non-functional. In pooled or multiplex formats, thereis likely to be selective enrichment of the cells with a non-functionalediting system since un-edited cells are not subjected to thedouble-strand DNA breaks that occur during editing. The strong selectionfor cells with non-functional nucleic acid-guided nuclease editingsystems leads to a disproportionate representation of non-edited cellsin the final cell population and compromises the integrity of librariesgenerated via multiplexed nuclease editing systems.

FIGS. 2B-2F depict improved protocols for performing nucleic acid-guidednuclease genome editing. FIG. 2B depicts a first embodiment 201 of animproved protocol for performing nucleic acid-guided nuclease genomeediting using an inducible promoter to drive expression of the gRNA. InFIG. 2B, a library or collection of editing vectors 202 is introduced203 (e.g., electroporated) into cultured cells 204 that comprise acoding sequence for a nuclease under the control of a constitutive orinducible promoter; however, the tightest regulation of the nucleicacid-guided nuclease system is achieved by using an inducible promoterto drive expression of the nuclease, thus it is preferred that aninducible promoter is used to drive transcription of the nuclease. Insome embodiments, the coding sequence for the nuclease is contained onan “engine plasmid” (most often along with, e.g., a selectable marker)that has already been transformed into the cells, although in otherembodiments the coding sequence for the nuclease may be integrated intothe genome of the cells. In yet other embodiments, the coding sequencefor the nuclease may be located on the editing vector (that is, acombined engine and editing vector). The editing vectors 202 comprise anediting sequence, which optionally includes a PAM-altering sequence(most often a sequence that disables the PAM at the target site in thegenome), a coding sequence for a gRNA under the control of an induciblepromoter, and a selectable marker.

Once the cells 204 have been transformed with the editing vectors, thecells are plated 205 on selective medium to select for cells that haveboth the engine and the editing vectors and grown until colonies 206form. Cells are then picked 207 from the colonies and grown overnightin, e.g., first 96-well plate 208 in medium that selects for both theengine and editing vectors. In a next step 209, the cells from the first96-well plate 208 are replicated into a second 96-well plate 210 intomedium containing an additional selective component such as, e.g.,arabinose, to drive strong induction of the λ red recombineering system(the homologous repair machinery). The inducible pBAD promoter—inducedby the presence of arabinose in the growth medium—controls the λ redrecombineering system and is described in relation to FIG. 11A below.Again, though a recombineering system is exemplified here in a bacterialediting system, recombineering systems generally are not needed ineukaryotic editing systems. After initial cell growth at 30° C., cuttingand editing of the cellular genome is induced by increasing thetemperature to 42° C. in the second 96-well plate 210 for, e.g.,one-half to approximately two hours (depending on cell type), toactivate the pL inducible promoters which drive transcription of thenuclease and the gRNA. Following induction of cutting and editing—e.g.,for approximately 2 hours—the temperature is returned to 30° C. to allowthe cells to recover.

In addition, in a step 211 cells from the first 96-well plate 208 arereplicated into a third 96-well plate 214 into medium that does notcontain the selective component that induces the homologous repairmachinery (here, arabinose, such that the λ Red recombineering system isnot activated). However, the third 96-well plate 214—aside from nothaving arabinose added to the growth medium—is subjected to the sameconditions as the second 96-well plate; that is, initial cell growth at30° C., increasing the temperature to 42° C. for, e.g., two hours toactivate the pL inducible promoter driving the expression of thenuclease and the gRNA, then reducing the temperature to 30° C. to allowthe cells to recover. As an alternative to 96-well plates 210 and 214,shown are two agar plates 212 and 216, where cells from plate 206 arearrayed. Plate 212 corresponds to the second 96-well plate 210,containing growth medium with arabinose, and plate 216 corresponds tothe third 96-well plate 214, containing growth medium without arabinose.Note that as an alternative to performing the method depicted in FIG. 2Bwith +/− arabinose, one may perform the method with −/+ temperatureinduction of cutting. For example, in a +/− temperature inductionexperiment, plate 210 would be the uninduced culture plate (no temp) andculture plate 214 would be the temperature-induced culture plate. Thesame overall interpretation applies; however, cut activity is isolatedto avoid having to deconvolute cut from paste.

Looking at the second and third 96-well plates (210 and 214,respectively) and at plates 212 and 216, colonies of cells can be seen.Looking first at second 96-well plate 210, there are two types ofcolonies: 226 (white wells) and 228 (hatched wells). Looking at third96-well plate 214, there are two types of colonies: 220 (black wells)and 226 (white wells). Replicate plating of cells from the first 96-wellplate into the second and third 96-well plates with differential mediaallows for functional deconvolution of the resulting cell populations.In third 96-well plate 214, cells were cultured in a medium withoutarabinose. With no arabinose, the λ Red recombineering system is notactive, and cells that have active gRNA and nuclease expression (inducedby increasing the temperature of the cultured cells to 42° C.) are notviable due to the double-strand cuts made by the active gRNA andnuclease yet without repair by the λ Red recombineering system. Thesecell colonies 222 are denoted by white wells. The cell colonies 220denoted by black wells represent cells that have inactive gRNAs, suchthat the genomes of cells in these colonies are not cut by the gRNA andnuclease complex. Inactive gRNAs occur at a frequency of approximately2-15% in a typical cell editing experiment, and typically are the resultof errors in the guide sequence portion (homology portion) of the gRNA.

In second 96-well plate 210, the cells were cultured in a medium witharabinose, thereby activating the λ Red recombineering system. Cellsthat have active gRNA and nuclease expression (induced by increasing thetemperature of the cultured cells to 42° C.) are cut, edited, and the λRed recombineering system repairs the double-strand DNA breaks and thusthese cells are viable. However, also viable are cells with inactivegRNAs (or less frequently, inactive nucleases). All viable colonies inplate 210 are denoted by hatched wells 228. Finally, cells that haveactive gRNAs but are not edited appropriately (due to, e.g., an inactiverecombineering system) are denoted by white wells 226. By comparing thesecond 96-well plate to the third 96-well plate, the function of thenucleic acid-guided nuclease system in each colony can be determined.For example, if colonies of cells grow without arabinose (plate 214,black wells 220) and with arabinose (plate 210, hatched wells 228), thecells must not have an active gRNA. If the cells had an active gRNA,they would not have been viable in medium without arabinose (e.g.,without an active λ Red recombineering system). If colonies of cellsfail to grow in medium without arabinose (plate 214, white wells 226)and do grow when arabinose is added to the culture medium (plate 210,hatched wells 228), the cells in these wells comprise an active nucleicacid-guided nuclease system—with active gRNA and nuclease components—andare very likely to have been properly edited. Finally, if the coloniesof cells fail to grow either without arabinose (plate 214, white wells226) or with arabinose (plate 210, white wells 226), the gRNA is active,but the cells are not able to repair the cut for some reason. Thus, themethod depicted in FIG. 2B allows for identification of edited cellcolonies via functional deconvolution of the various components of theediting “machinery” in one experiment.

In plates 212 and 216 the phenotypic readout is the same as in 96-wellplates 210 and 214. Like 96-well plate 210, the medium in culture dish212 contains arabinose, and like 96-well plate 214, the medium inculture dish 216 does not contain arabinose. Thus, in culture dish 216cells with inactive gRNAs grow, as there is no edit (double-strandbreak) to repair. However, in cells with an active gRNA, editing takesplace but there is no active repair machinery to repair the edits, andthe cells are not viable and do not form colonies. In culture dish 212that contains arabinose, cells that have inactive gRNAs form colonies,as do cells that have active gRNAs but are properly edited. Cells thatare not viable for whatever reason do not form colonies. As with 96-wellplates 210 and 214, comparison of culture dishes 212 and 214 allow oneto deconvolute the various components of the editing machinery. If cellsgrow on both culture dishes 212 and 214, the gRNA is likely inactive. Ifcells grow on culture dish 212 but not on culture dish 214, the gRNA islikely active and proper editing has taken place. If cells fail to growon either culture dish 212 or 214, the cells likely have an active gRNAbut the edit is not repaired properly.

Thus, the method 201 depicted in FIG. 2B allows for identification ofcells with nonfunctional gRNAs that, due to the lack of double-strandDNA breaks, have a growth advantage. Most importantly, the method 201depicted in FIG. 2B allows for identification of cells that have beenproperly edited. An aliquot of the colonies 228 from either second96-well plate 210 or agar plate 212—the colonies confirmed to have anactive gRNA—can be picked and sequenced to confirm editing. 96-wellplate 210 or agar plate 212 may be retained so that once proper editingis confirmed by, e.g., sequencing, one can go back to plate 210 or 212and retrieve the properly-edited cells. Plates 210 and 212 may bereferred to, e.g., as “cell hotels” or “cell repositories.” Note thatthe method 201 depicted in FIG. 2B first substantially or largelysingulates the cells by plating them on selective medium at anappropriate dilution such that single cells form single colonies. Again,singulation or substantial singulation overcomes growth the bias that ischaracteristic of unedited cells. The method then allows for functionaldeconvolution of the different colonies by replica plating colonies in96-well microtiter plates under different section media to allowpositive identification of edited colonies. A specific protocol for thisexemplary method is described in Example 6 below.

FIG. 2C depicts a second exemplary embodiment of an improved protocol230 for performing nucleic acid-guided nuclease genome editing using aninducible promoter to drive expression of the gRNA, and, preferably, thenuclease as well. In FIG. 2C as in FIG. 2B, a library or collection ofediting vectors 202 is introduced 203 (e.g., electroporated) intocultured cells 204 that comprise a coding sequence for a nuclease underthe control of a constitutive or inducible promoter; however, thetightest regulation of the nucleic acid-guided nuclease system isachieved by using an inducible promoter to drive expression of thenuclease and thus is preferred. Also like FIG. 2B, in some embodiments,the coding sequence for the nuclease is contained on an “engine plasmid”(most often along with, e.g., a selectable marker) that has already beentransformed into the cells, although in other embodiments, the codingsequence for the nuclease may be integrated into the genome of thecells. In yet other embodiments, the coding sequence for the nucleasemay be located on the editing vector (that is, a combined engine andediting vector). The editing vectors 202 comprise an editing sequencewith a desired edit vis-à-vis an endogenous nucleic acid sequence in thecell along with a PAM-altering sequence (most often a sequence thatdisables the PAM at the target site in the genome), a coding sequencefor a gRNA under the control of, preferably, an inducible promoter, anda selectable marker.

Once the cells 204 have been transformed with the editing vectors, thecells are plated 235 on selective medium on substrate or plate 236 toselect for cells that have both the engine and the editing vectors. Thecells are diluted before plating such that the cells are substantiallyor largely singulated—separated enough so that they and the coloniesthey form are separated from other cell colonies—and the cells are thengrown 237 on plate or substrate 236 until colonies 238 begin to form.The cells are allowed to grow at, e.g., 30° C. for, e.g., between 2 and150, or between 5 and 120, or between 10 and 50 doublings, establishingclonal colonies. This initial growth of cells is to accumulate enoughclonal cells in a colony to survive induction of editing. Once coloniesare established, cutting and editing of the cellular genome is inducedby inducing or activating the promoters driving one or both of the gRNAand nuclease, and the λ Red recombineering system (if present). If the λRed recombineering system is present and under the control of aninducible promoter, preferably this inducible promoter is different fromthe inducible promoter driving transcription of the gRNA and nucleaseand is activated (induced) before induction of the gRNA and nuclease.The λ Red recombineering system works as the “band aid” or repair systemfor double-strand breaks in bacteria, and in some species of bacteriamust be present for the double-strand breaks that occur during editingto resolve. The λ Red recombineering system may be under the control of,e.g., a pBAD promoter. The pBAD promoter, like the pL promoter, is aninducible promoter; however, the pBAD promoter is regulated (induced) bythe addition of arabinose to the growth medium. Thus, if there isarabinose contained in the selective medium of substrate or plate 236,the λ Red recombineering system will be activated when the cells aregrown 237. As for induction of editing 239, if transcription of the gRNAand nuclease are both under control of the pL promoter, transcription ofthe gRNA and nuclease is induced by increasing the temperature to 42° C.for, e.g., a half-hour to two hours (or more, depending on the celltype), which activates the pL inducible promoter. Following induction ofcutting and editing and a two-hour 42° C. incubation, the temperature isreturned to 30° C. to allow the cells to recover and to disable the pLpromoter system.

Once the cells have recovered and are growing at 30° C., the cells aregrown on substrate or plate 236 into colonies of terminal size 244; thatis, the colonies arising from the substantially or largely singulatedcells are grown into colonies to a point where cell growth has peakedand becomes normalized (e.g., saturated) for both edited and uneditedcells. As described supra, normalization occurs as the nutrients in themedium around a growing cell colony are depleted and/or cell growthfills the microwells or is otherwise constrained; that is, the cells arein senescence. The terminal-size colonies are then pooled 241 by, e.g.,scraping the colonies off the substrate or plates (or by, e.g., flushingthe clonal cell colonies from microwells in a solid wall device) to pool280 the cells from the normalized cell colonies. Note that the method240 illustrated and described for FIG. 2C utilizes singulation orsubstantial singulation, initial growth, induction, normalization andpooling of the resulting cell colonies. Again, because singulation orsubstantial singulation overcomes growth bias from unedited cells orcells exhibiting fitness effects as the result of edits made, thecombination of singulation or substantial singulation, initial growth,induction of editing, and normalization enriches the total population ofcells with cells that have been edited. Note, however, unlike themethods 201, 250 depicted in FIG. 2B and FIG. 2D respectively, method240 in FIG. 2C takes no steps to deconvolute the editing “machinery.”

FIG. 2D depicts a third exemplary embodiment of an improved protocol 250for performing nucleic acid-guided nuclease genome editing using aninducible promoter to drive expression of the gRNA, and in someembodiments and preferably, the nuclease as well. FIG. 2D depicts aprotocol 250 for activity-based error correction and re-arraying. InFIG. 2D as in FIGS. 2B and 2C, a library or collection of editingvectors 202 is introduced 203 (e.g., electroporated) into cultured cells204 that comprise a coding sequence for a nuclease under the control ofa constitutive or inducible promoter (preferably inducible), eithercontained on an “engine plasmid” (e.g., along with, e.g., a selectablemarker) that has already been transformed into the cells, or integratedinto the genome of the cells. Alternatively, the coding sequence for thenuclease may be located on the editing vector. The editing vectors 202comprise an editing sequence, a PAM-altering sequence (most often asequence that disables the PAM at the target site in the genome), acoding sequence for a gRNA under the control of, preferably, aninducible promoter, and a selectable marker.

Once the cells have been transformed with the editing vectors, the cellsare diluted and plated 205 on selective medium 206 to select for cellsthat have both the engine and the editing vectors (e.g., medium withchloramphenicol and carbenicillin) and grown until colonies form 221,again in a process that substantially or largely singulates the cells.Cells are then picked 207 from the colonies 221 and grown overnight in,e.g., first 96-well plate 208 in medium that does not comprise aselective medium. In a next step, the cells from the first 96-well plateare replicated 211 into a second 96-well plate 214 in medium withoutarabinose, resulting in wells with colonies (black wells 220), and wellswithout cells colonies (white wells 222). This process identifies cellswith active gRNAs, as cells with active gRNAs will not survive becausewithout arabinose the λ Red recombineering system is not active torepair the cuts in the genome made by the gRNA. Thus, the wells 222likely denote wells with active gRNAs. The colonies that do grow in thissecond 96-well plate 214 likely have inactive gRNAs, and thus there isno cut to repair and the cells remain viable (denoted by black wells220).

Once the cells with the active gRNAs are identified (wells 222), thesecells are then “cherry-picked” 213 from the first 96-well plate andarrayed into a third 96-well plate 252 to be kept as a cell repositoryor “cell hotel.” All colonies in the third 96-well plate 252 are cellsthat have been identified as likely having active gRNAs 222. With thiscell repository 252, aliquots of these “cherry-picked” cells (e.g., herecells with active gRNAs) can then be arrayed 255 into a fourth 96-wellplate 254 in medium with arabinose. After an initial growth period, thecells in this fourth 96-well plate 254 are subjected to cutting andediting conditions, e.g., increasing the temperature to 42° C. for,e.g., two hours, to induce the pL inducible promoter driving theexpression of the nuclease and the gRNA. Once the cutting and editingprocesses take place, colonies containing cells that have been properlyedited can be identified by monitoring growth of the colonies andselecting slow-growing colonies or by targeted or whole genomesequencing taking cells from plate 254. Once cells with desired editsare identified, the cells with the desired edits can be retrieved from96-well plate 252 (e.g., the cell repository or “cell hotel”).Alternatively, the cells colonies with the putatively active gRNAs onplate 252 can be pooled 253 into a mixed cell culture 256 and eitheranalyzed or subjected to an additional round of editing.

FIG. 2E depicts yet another exemplary embodiment of an improved protocol270 for performing nucleic acid-guided nuclease genome editing using aninducible promoter to drive expression of one or more of the gRNA and/orthe nuclease. The protocol 270 in FIG. 2D does not entail functionaldeconvolution of the editing “machinery” of the cells but depicts aprotocol for high-throughput screening using colony morphology toidentify edited cells. Again, in edited cells, cell viability iscompromised in the period after editing is induced. The present methodtakes advantage of the growth lag in colonies of edited cells toidentify edited cells. In some embodiments, the colony size of theedited cells is 20% smaller than colonies of non-edited cells. In someaspects, the colony size of the edited cells is 30%, 40%, 50%, 60%, 70%,80% or 90% smaller than the colonies of non-edited cells. In manyembodiments, the colony size of the edited cells is 30-80% smaller thancolonies of non-edited cells, and in some embodiments, the colony sizeof the edited cells is 40-70% smaller than colonies of non-edited cells.

In FIG. 2E as in FIGS. 2B-2D, a library or collection of editing vectors202 is introduced 203 (e.g., electroporated) into cultured cells 204that comprise a coding sequence for a nuclease under the control of aconstitutive or inducible promoter (preferably an inducible promoter),contained 1) on an “engine plasmid” (most often along with a selectablemarker) that has already been transformed into the cells; 2) integratedinto the genome of the cells being transformed; or 3) the codingsequence for the nuclease may be located on the editing vector. Theediting vectors 202 comprise an editing sequence and optionally includea PAM-altering sequence (e.g., a sequence that disables the PAM at thetarget site in the genome), a coding sequence for a gRNA under thecontrol of, preferably, an inducible promoter, and a selectable marker.In many embodiments, an inducible promoter is included in the editingvector backbone, and an editing cassette is inserted 3′ of the induciblepromoter where the editing cassette comprises, from 5′ to 3′: a codingsequence for a gRNA, and a donor DNA comprising a desired edit for atarget sequence and the PAM-altering sequence (e.g., a CREATE cassette).Note again, that transcription of one or both of the gRNA and nucleasemust be under the control of an inducible promoter. It is preferred,however, that transcription of both the gRNA and the nuclease is underthe control of an inducible promoter.

At step 271, the transformed cells are diluted and plated (e.g.,substantially or largely singulated) onto selective medium 272 thatselects for both the engine and editing vectors (e.g., medium containingboth chloramphenicol and carbenicillin) and further contains arabinoseso as to activate the λ Red recombineering system. Once plated, thecells are grown 273 at 30° C. for 6-8 hours so that the cells initiallyestablish colonies, then the temperature is increased to 42° C. for,e.g., on-half to two hours to induce expression of the nuclease and gRNAfor cutting and editing. Once sufficient time for editing has takenplace, the temperature is returned to 30° C. so that the cells grow tore-establish colonies on plate 272. Once colonies appear, there arelarge 278 and small 276 colonies. The colonies with small size 276 areindicative of an active gRNA and likely to have been edited as thedouble-strand cuts caused by active editing are largely toxic to thecells, resulting both in cell death in the edited colonies as well as alag in growth for the edited cells that do survive but must repair andrecover following editing. The small colonies (edited cells) are cherrypicked 277 and are arrayed on a 96-well plate 282. Cells in the 96-wellplate 282 can be cultured, and aliquots from this 96-well plate 282 canbe sequenced and colonies with desired edits identified. This 96-wellplate may be kept as a cell hotel or cell repository, and once cellsthat have been properly edited are identified, one can retrieve thecells with the desired edit from “cell hotel” 96-well plate 282.

Alternatively, small colonies 276 may be picked and pooled 275 foradditional rounds of editing since the edited cells have been selectedin the first round of editing and likely comprise edits from the firstround. Note again, that this method does not provide functionaldeconvolution of the editing machinery; however, the method depicted inFIG. 2E employs both singulation or substantial singulation and cherrypicking and thus enables for a high-throughput diagnostic method toidentify cells based on the colony size morphology that have a highlikelihood of being edited, and once identified, the edited cellpopulation can be enriched. Screening out a large proportion of thecells with non-functional gRNAs allows for identification of editedcells more readily. It has been determined that removing growth ratebias via singulation or substantial singulation improves the observedediting efficiency by up to 2×, 3×, 4× or more over conventionalmethods, and further that cherry-picking colonies using the methodsdescribed herein increases by 1.5×, 1.75×, 2.0×, or 2.5× or more theobserved editing efficiency of singulation. Thus, the combination ofsubstantial singulation and cherry picking improve observed editingefficiency by 8x over conventional methods. Example 9 below providesmaterials and methods for this embodiment.

While the method for screening for edited cells using cell growth as aproxy for editing has been described herein in the context of measuringcolony size of cell colonies on an agar plate, the optical density (OD)of growing cell colonies, such as in a microtiter plate or in a seriesof tubes may be measured instead. Moreover, other cell growth parameterscan be measured in addition to or instead of cell colony size or OD. Forexample, spectroscopy using visible, UV, or near infrared (NIR) lightallows monitoring the concentration of nutrients and/or wastes in thecell culture. Additionally, spectroscopic measurements may be used toquantify multiple chemical species simultaneously. Nonsymmetric chemicalspecies may be quantified by identification of characteristic absorbancefeatures in the NIR. Conversely, symmetric chemical species can bereadily quantified using Raman spectroscopy. Many critical metabolites,such as glucose, glutamine, ammonia, and lactate have distinct spectralfeatures in the IR, such that they may be easily quantified. The amountand frequencies of light absorbed by the sample can be correlated to thetype and concentration of chemical species present in the sample. Eachof these measurement types provides specific advantages. FT-NIR providesthe greatest light penetration depth and so can be used for thickersample so that they provide a higher degree of light scattering.FT-mid-IR (MIR) provides information that is more easily discernible asbeing specific for certain analytes as these wavelengths are closer tothe fundamental IR absorptions. FT-Raman is advantageous when theinterference due to water is to be minimized. Other spectral propertiescan be measured via, e.g., dielectric impedance spectroscopy, visiblyfluorescence, fluorescence polarization, or luminescence. Additionally,sensors for measuring, e.g., dissolved oxygen, carbon dioxide, pH,and/or conductivity may be used to assess the rate of cell growth.

FIG. 2F depicts additional detail of the exemplary embodiment shown inFIG. 2E, using a specific example based on experiments performed (seeExample 1, infra). FIG. 2F shows high-throughput screening 290 usingcolony morphology to identify or cherry pick edited cells. As describedabove, in edited cells cell viability is compromised in the period afterediting is induced. The present method takes advantage of the growth lagin colonies of edited cells to identify edited cells. In FIG. 2F,transformed cells are diluted and plated on medium containing arabinoseand grown for a period of time at, e.g., 30° C. Editing is initiated by,e.g., raising the temperature to 42° C. for a period of time, then thetemperature is lowered to 30° C. (e.g., the transformed cells aresubstantially or largely singulated). Colonies are allowed to grow andboth small 276 and large 278 colonies result. Colonies from this plateare picked 291 and arrayed on a second plate 292 containing selectivemedium, e.g., a medium to select for successful editing of galK,resulting in white (versus red) colonies when plated on MacConkey agarsupplemented with galactose as the sole carbon source. Note that pickingsmall colonies 293 from the first plate results primarily in editedcells 296 (white colonies, shown here as open circles) and—at a muchlower frequency—some cells in which the gRNA is inactive 294 (redcolonies, shown here as filled-in circles). Confirmation of colonies inwhich the gRNA is inactive is shown by picking 295 large colonies 278from the first plate and plating them on the second plate (colonies 298)where these cells result in red colonies when grown on MacConkey agarsupplemented with galactose as the sole carbon source thus confirming aninactive gRNA or other part of the editing machinery. Thus, using smalland large colony morphology as a proxy for edited and non-edited cells,respectively, provides a high throughput and facile screening method foredited cells. Note that the methods depicted in FIGS. 2E and 2F employboth singulation or substantial singulation and cherry-pickingstrategies.

The exemplary workflows described herein employ the concept ofsingulation. Singulation overcomes the growth bias in favor of uneditedcells, thus allowing edited cells “equal billing” with unedited cells.Further, the methods take advantage of a tightly-regulated induciblesystem to screen edited cells from non-edited cells (both cells wherethe gRNA is non-functional, and cells where the gRNAs are functional butsome component of the nucleic acid-guided nuclease system is notfunctional). Screening may be performed using replica plates andidentifying “escapees” or cells in which the gRNA is non-functional,screening may be performed by taking advantage of the growth lag ofedited cells in comparison to non-edited cells, or enrichment can beperformed by using a combination of singulation or substantialsingulation, initial growth, induction of editing and normalization. Theresult of the methods is that even in nucleic acid-guided nucleasesystems where editing is not optimal (such as in systems wherenon-canonical PAMs are targeted), there is an increase in the observedediting efficiency; that is, edited cells can be identified even in alarge background of unedited cells.

Note that the methods depicted in FIGS. 2B-2F show cell colonysingulation, initial growth, and induction of editing on solid medium incell culture dishes or in 96-well plates. It should be recognized by oneof ordinary skill in the art given the discussion herein thatsingulation, initial growth, and induction of editing can be performedin other formats, such as, e.g., in the solid wall devices described inrelation to FIGS. 3A-3F and 4A-4CC, or as described in U.S. Ser. No.62/735,365, entitled “Detection of Nuclease Edited Sequences inAutomated Modules and Systems”, filed 24 Sep. 2018, and U.S. Ser. No.62/781,112, entitled “Improved Detection of Nuclease Edited Sequences inAutomated Modules and Systems,” filed 18 Dec. 2018, which includedescriptions of singulation or substantial singulation by isolatingcells on functionalized islands, singulation or substantial singulationwithin aqueous droplets carried in a hydrophobic carrier fluid or GelBeads-in-Emulsion (GEMs, see, e.g., 10× Genomics, Pleasanton, Calif.),or singulation or substantial singulation within a polymerized alginatescaffold (for this embodiment of singulation, also see U.S. Ser. No.62/769,805, entitled “Improved Detection of Nuclease Edited Sequences inAutomated Modules and Instruments via Bulk Cell Culture”, filed 20 Nov.2018).

Exemplary Modules for Editing, Enrichment, and Selection of Edited Cells

The instruments, methods, and modules described herein enable enhancedobserved editing efficiency of nucleic acid-guided nuclease editingmethods as the result of singulation or substantial singulation, initialcell growth, induction of editing, and normalization. The combination ofthe singulation or substantial singulation, initial growth, induction ofediting and normalization processes overcomes the growth bias in favorof unedited cells—and the fitness effects of editing (includingdifferential editing rates)—thus allowing all cells “equal billing” withone another. The result of the instruments, modules, and methodsdescribed herein is that even in nucleic acid-guided nuclease systemswhere editing is not optimal (such as in systems where non-canonicalPAMs are targeted), there is an increase in the observed editingefficiency; that is, edited cells can be identified even in a largebackground of unedited cells. Observed editing efficiency can beimproved up to 80% or more.

FIG. 3A depicts a solid wall device 350 and a workflow for singulatingcells in microwells in the solid wall device, where in this workflow oneor both—preferably both—of the gRNA and nuclease are under the controlof an inducible promoter. At the top left of the figure (i), there isdepicted solid wall device 350 with microwells 352. A section 354 ofsubstrate 350 is shown at (ii), also depicting microwells 352. At (iii),a side cross-section of solid wall device 350 is shown, and microwells352 have been loaded, where, in this embodiment, Poisson or substantialPoisson loading has taken place; that is, each microwell has one or nocells, and the likelihood that any one microwell has more than one cellis low. At (iv), workflow 340 is illustrated where substrate 350 havingmicrowells 352 shows microwells 356 with one cell per microwell,microwells 357 with no cells in the microwells, and one microwell 360with two cells in the microwell. In step 351, the cells in themicrowells are allowed to double approximately 2-150 times to formclonal colonies (v), then editing is induced 353 by heating thesubstrate (e.g., for temperature-induced editing) or flowing chemicalsunder or over the substrate (e.g., sugars, antibiotics forchemical-induced editing) or by moving the solid wall device to adifferent medium, particularly facile if the solid wall device is placedon a membrane which forms the bottom of microwells 352 (membrane notshown).

After induction of editing 353, many cells in the colonies of cells thathave been edited die as a result of the double-strand cuts caused byactive editing and there is a lag in growth for the edited cells that dosurvive but must repair and recover following editing (microwells 358),where cells that do not undergo editing thrive (microwells 359) (vi).All cells are allowed to continue grow 355 to establish colonies andnormalize, where the colonies of edited cells in microwells 358 catch upin size and/or cell number with the cells in microwells 359 that do notundergo editing (vii). Once the cell colonies are normalized, eitherpooling 360 of all cells in the microwells can take place, in which casethe cells are enriched for edited cells by eliminating the bias fromnon-editing cells and fitness effects from editing; alternatively,colony growth in the microwells is monitored after editing, and slowgrowing colonies (e.g., the cells in microwells 358) are identified andselected 361 (e.g., “cherry picked”) resulting in even greaterenrichment of edited cells.

In growing the cells, the medium used will depend, of course, on thetype of cells being edited—e.g., bacterial, yeast or mammalian. Forexample, medium for bacterial growth includes LB, SOC, M9 Minimalmedium, and Magic medium; medium for yeast cell growth includes TPD,YPG, YPAD, and synthetic minimal medium; and medium for mammalian cellgrowth includes MEM, DMEM, IMDM, RPMI, and Hanks. For culture ofadherent cells, cells may be disposed on beads or another type ofscaffold suspended in medium. Most normal mammalian tissue-derivedcells—except those derived from the hematopoietic system—are anchoragedependent and need a surface or cell culture support for normalproliferation. In the rotating growth vial described herein,microcarrier technology is leveraged. Microcarriers of particular usetypically have a diameter of 100-300 μm and have a density slightlygreater than that of the culture medium (thus facilitating an easyseparation of cells and medium for, e.g., medium exchange) yet thedensity must also be sufficiently low to allow complete suspension ofthe carriers at a minimum stirring rate in order to avoid hydrodynamicdamage to the cells. Many different types of microcarriers areavailable, and different microcarriers are optimized for different typesof cells. There are positively charged carriers, such as Cytodex 1(dextran-based, GE Healthcare), DE-52 (cellulose-based, Sigma-AldrichLabware), DE-53 (cellulose-based, Sigma-Aldrich Labware), HLX 11-170(polystyrene-based); collagen or ECM (extracellular matrix)-coatedcarriers, such as Cytodex 3 (dextran-based, GE Healthcare) or HyQ-spherePro-F 102-4 (polystyrene-based, Thermo Scientific); non-chargedcarriers, like HyQspheres P 102-4 (Thermo Scientific); or macroporouscarriers based on gelatin (Cultisphere, Percell Biolytica) or cellulose(Cytopore, GE Healthcare).

FIG. 3B depicts a solid wall device 350 and a workflow for substantiallysingulating cells in microwells in a solid wall device, where in thisworkflow (as in the workflow depicted in FIG. 3A) one or both—preferablyboth—of the gRNA and nuclease is under the control of an induciblepromoter. At the top left of the figure (i), there is depicted solidwall device 350 with microwells 352. A section 354 of substrate 350 isshown at (ii), also depicting microwells 352. At (iii), a sidecross-section of solid wall device 350 is shown, and microwells 352 havebeen loaded, where, in this embodiment, substantial Poisson loading hastaken place; that is, some microwells 357 have no cells, and somemicrowells 376, 378 have a few cells. In FIG. 3B, cells with activegRNAs are shown as solid circles, and cells with inactive gRNAs areshown as open circles. At (iv), workflow 370 is illustrated wheresubstrate 350 having microwells 352 shows three microwells 376 withseveral cells all with active gRNAs, microwell 357 with no cells, andtwo microwells 378 with some cells having active gRNAs and some cellshaving inactive gRNAs. In step 371, the cells in the microwells areallowed to double approximately 2-150 times to form clonal colonies (v),then editing is induced 373 by heating the substrate (e.g., fortemperature-induced editing) or flowing chemicals under or over thesubstrate (e.g., sugars, antibiotics for chemical-induced editing) or bymoving the solid wall device to a different medium, particularly facileif the solid wall device is placed on a membrane which forms the bottomof microwells 352.

After induction of editing 373, many cells in the colonies of cells thathave been edited die as a result of the double-strand cuts caused byactive editing and there is a lag in growth for the edited cells that dosurvive but must repair and recover following editing (microwells 376),where cells that do not undergo editing thrive (microwells 378) (vi).Thus, in microwells 376 where only cells with active gRNAs reside (cellsdepicted by solid circles), most cells die off; however, in microwells378 containing cells with inactive gRNAs (cells depicted by opencircles), cells continue to grow and are not impacted by active editing.The cells in each microwell (376 and 378) are allowed to grow 375 tocontinue to establish colonies and normalize, where the colonies ofedited cells in microwells 376 catch up in size and/or cell number withthe unedited cells in microwells 379 that do not undergo editing (vii).Note that in this workflow 370, the colonies of cells in the microwellsare not clonal; that is, not all cells in a well arise from a singlecell. Instead, the cell colonies in the well may be mixed colonies,arising in many wells from two to several different cells. Once the cellcolonies are normalized, either pooling 390 of all cells in themicrowells can take place, in which case the cells are enriched foredited cells by eliminating the bias from non-editing cells and fitnesseffects from editing; alternatively, colony growth in the microwells ismonitored after editing, and slow growing colonies (e.g., the cells inmicrowells 376) are identified and selected 391 (e.g., “cherry picked”)resulting in even greater enrichment of edited cells.

FIG. 3C is a photograph of one embodiment of a solid wall substratecomprising microwells for singulating cells. As can be seen from thephoto, the solid wall substrate is a perforated disk of metal and isapproximately 2 inches (˜47 mm) in diameter. The perforated disk seen inthis photograph is fabricated 316 stainless steel, where theperforations form the walls of the microwells, and a filter or membraneis used to form the bottom of the microwells. Use of a filter ormembrane (such as a 0.22 μm PVDF Duropore™ woven membrane filter) allowsfor medium and/or nutrients to enter the microwells but prevents thecells from flowing down and out of the microwells. Filter or membranemembers that may be used in the solid wall singulation or substantialsingulation/initial growth/induction of editing and normalization orcherry picking devices and modules are those that are solvent resistant,are contamination free during filtration, and are able to retain thetypes and sizes of cells of interest. For example, in order to retainsmall cell types such as bacterial cells, pore sizes can be as low as0.10 μm, however for other cell types, the pore sizes can be as high as0.5 μm. Indeed, the pore sizes useful in the cell concentrationdevice/module include filters with sizes from 0.10 μm, 0.11 μm, 0.12 μm,0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.20 μm,0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm,0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm,0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm,0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. Thefilters may be fabricated from any suitable material including cellulosemixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, or glass fiber.

In the photograph shown in FIG. 3C, the perforations are approximately150 μm-200 μm in diameter, resulting in the microwells having a volumeof approximately 2.5 nl, with a total of approximately 30,000microwells. The distance between the microwells is approximately 279 nmcenter-to-center. Though here the microwells have a volume ofapproximately 2.5 nl, the volume of the microwells may be from 1 to 25nl, or preferably from 2 to 10 nl, and even more preferably from 2 to 4nl. The preferred size/volume of the microwells will depend of cell type(e.g., bacterial, yeast, mammalian). The perforated disk shown here ismade of 316 stainless steel; however other bio-compatible metals andmaterials may be used. The solid wall device may be disposable or it maybe reusable. The solid wall device shown in FIG. 3C is round, but can beof any shape, for example, square, rectangular, oval, etc. (See, e.g.,FIG. 4A.) Round perforated disks are useful if petri dishes are used tosupply the solid wall module with nutrients via solid medium in, e.g., apetri or other cell culture dish. The filters used to form the bottom ofthe microwells of the solid wall device include 0.22 μm PVDF Duropore™woven membrane filters. Further, though a 2-inch (—47 mm) diameterperforated disk is shown, the perforated disks may be smaller or largeras desired and the configuration of the solid wall module will depend onhow nutrients are supplied to the solid wall module, and how mediaexchange is performed. For example, see the perforated member of FIG. 4Aand the embodiments of a solid wall module in FIGS. 4F-4BB.

FIGS. 3D-3F are photographs of E. coli cells substantially or largelysingulated via Poisson or substantial Poisson distribution in microwellsor perforations in a perforated disk with a membrane bottom at low,medium and high magnification, respectively. FIG. 3D shows digitalgrowth at low magnification where the darker microwells are microwellswhere cells are growing. FIG. 3E is a top view of microwells in aperforated disk where the darker microwells are microwells where cellsare growing. FIG. 3F is a photograph of microwells where the membrane(e.g., the permeable membrane that forms the bottom of the microwells)has been removed, where unpatterned (smooth) microwells are microwellswhere cells are not growing, and microwells with irregularpigment/patterned are microwells where cells are growing, and, in thisphotograph, have filled the microwells in which they are growing. Inthese photographs, a 0.2 μm filter (membrane) was swaged against theperforated disk under high pressure such as the round perforated diskdepicted in FIG. 3C. The perforated disk formed the walls of themicrowells, and the 0.2 μm filter formed the bottom of the microwells.To load the perforated disk+filter, the E. coli cells were pulled intothe microwells using a vacuum (see Example 6 for methods). Theperforated disk+filter was then placed on an LB agar plate membrane-sidedown, and the cells were grown overnight at 30° C., then two days atroom temperature. The membrane was removed and the bottomless microwellswere photographed by light microscopy. Note the ease with whichdifferent selective media can be used to select for certain cellphenotypes; that is, one need only transfer the perforated disk+filterto a different plate or petri dish comprising a desired selectivemedium. Generally the number of cells loaded into a singulation deviceor singulation assembly ranges from between approximately 0.1× to 2.5×the number of perforations or microwells, or from between approximately0.3× to 2.0× the number of perforations or microwells, or from betweenapproximately 0.5× to 1.5× the number of perforations or microwells.

FIGS. 4A through 4BB depict various components of different embodimentsand components of a solid wall singulation or substantial singulation,growth, induction of editing and either normalization or cherry pickingmodule (“solid wall isolation/induction/normalization module” or“SWIIN”) suitable for singulating (or substantially singulating) cellsof all types, growing cells for an initial, e.g., 2-150 rounds of celldivision, inducing editing, and either normalizing or cherry picking theresulting cell colonies. The SWIIN modules presented may be stand-alonedevices, or, often, one module in an automated multi-module cellprocessing instrument. FIG. 4A is a representation of a perforated metalsheet or perforated member 401 that is generally rectangular in shape.The perforations are not represented at scale. As with the perforateddisk described in relation to FIGS. 3C-3F, perforated member 401 isfabricated from 316 stainless steel, where the perforations form thewalls of microwells, and a filter or membrane (not seen in this FIG. 4A)is used to form the bottom of the microwells.

In the scanning electromicrograph shown in FIG. 4B the perforations(microwells) are approximately 150 μm-200 μm in diameter, and theperforated member is approximately 125 deep, resulting in microwellshaving a volume of approximately 2.5 nl, with a total of approximately200,000 microwells. The distance between the microwells is approximately279 μm center-to-center. Though here the microwells have a volume ofapproximately 2.5 nl, the volume of the microwells may be from 1 to 25nl, or preferably from 2 to 10 nl, and even more preferably from 2 to 4nl. The perforated member pictured in FIG. 4A is approximately 14 cmlong and 10 cm (140 mm×100 mm) wide; however, smaller perforated members(such as shown in FIG. 3C) or larger perforated members may be useddepending on the density of microwells in the perforated member and thenumber of microwells or partitions required. For example, the larger theplexity (e.g., complexity) of the library being used to edit apopulation of cells, a larger number of microwells or partitions ispreferred. If, for example, a 10,000-plex library is used to edit apopulation of cells, a perforated member with 200,000 microwells orpartitions would be more than adequate; however, if a 50,000-plexlibrary is used to edit a population of cells, a perforated member (ortwo or more members) with a total of 400,000 or more microwells orpartitions may be preferred. Generally the number of cells loaded into asingulation device or singulation assembly ranges from betweenapproximately 0.1× to 2.5× the number of perforations or microwells, orfrom between approximately 0.3× to 2.0× the number of perforations ormicrowells, or from between approximately 0.5× to 1.5× the number ofperforations or microwells; thus, the number of cells loaded onto aperforated member comprising approximately 200,000 perforations wouldrange from about 20,000 cells to about 500,000 cells, or from about60,000 cells to about 400,000 cells, or from about 100,000 cells toabout 300,000 cells. The preferred size/volume of the microwells willdepend on the cell type (e.g., archeal, bacterial, yeast, non-mammalianeukaryotic, and/or mammalian being edited). The perforated member shownhere is made of 316 stainless steel; however other bio-compatible metalsand materials may be used, such as titanium, cobalt-based alloys, andceramics. The SWIIN may be disposable or it may be reusable. If reused,the SWIIN may be heated to 55° C. or greater to sterilize the SWIINalternatively, antibiotics may be flushed through the SWIIN.

FIGS. 4B-4E are scanning electromicrographs of a portion of a perforatedmember 401 (FIG. 4B), a close up of one microwell 402 without (FIG. 4C)and with (FIGS. 4D and 4E) a filter membrane forming the bottom of themicrowell 402. FIG. 4B shows perforated member 401 and 30 or somicrowells 402. FIG. 4C also shows perforated member 401 and a singlemicrowell 402, where this microwell is approximately 172 μm in diameter.FIG. 4D shows perforated member 401 and approximately 8 microwells 402,each of which has a portion of a filter or membrane 403 forming thebottom of the microwell 402. FIG. 4E is a higher magnificationmicrograph of one of the microwells 402 from the perforated member 401shown in FIG. 4D. As described above in relation to FIGS. 3C-3F, use ofa filter or membrane (such as a 0.22 μm PVDF Duropore™ woven membranefilter) allows for medium and/or nutrients to enter the microwells butprevents cells from flowing down and out of the microwells. Filter ormembrane members that may be used to form the bottom of the microwellsof a perforated member in a solid wall singulation or substantialsingulation/initial growth/induction of editing and normalization/cherrypicking module are those that are solvent resistant, are contaminationfree during filtration, do not tear under pressures required to exchangemedia and load cells, and are able to retain the types and sizes ofcells of interest. For example, in order to retain small cell types suchas bacterial cells, pore sizes can be as low as 0.2 μm, however forother cell types, the pore sizes can be as high as 0.5 μm or larger. Thefilters may be fabricated from any suitable material including cellulosemixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, or glass fiber. The perforatedmember 401 and filter 403 are swaged together; that is, the perforatedmember 401 and filter 403 are pressed together under high pressure(e.g., 20 kpsi). In some embodiments, the holes or partitions in theperforated member are etched with a slight taper and the filter materialis pressed into the opening then relaxed to form an effective swage,allowing for an adhesive-free coupling. As an alternative, an adhesivecould be employed.

FIG. 4F depicts one embodiment of a singulation assembly 420 a from atop perspective view, which presents the “retentate side” of singulationassembly 420 a. As used herein a “singulation assembly” comprises aretentate member 404 as a top member, a permeate member 408 as a bottommember, with a gasket 416 surrounding a perforated member 401 and afilter 403, where gasket 416, perforated member 401 and filter 403 aresandwiched between retentate member 404 and permeate member 408.Retentate and permeate members 404 and 408, respectively, in theembodiments exemplified in FIGS. 4F-4Q and 4Y are transparent, and areapproximately 200 mm long, 130 mm wide, and 4 mm thick, though in otherembodiments, the retentate and permeate members can be from 75 mm to 350mm in length, or from 100 mm to 300 mm in length, or from 150 mm to 250mm in length; from 50 mm to 250 mm in width, or from 75 mm to 200 mm inwidth, or from 100 mm to 150 mm in width; and from 2 mm to 15 mm inthickness, or from 4 mm to 10 mm in thickness, or from 5 mm to 8 mm inthickness. In the embodiments depicted in FIGS. 4F-4BB, the retentatemembers are fabricated from PMMA (poly(methyl methacrylate); however,other materials may be used, including polycarbonate, cyclic olefinco-polymer (COC), glass, polyvinyl chloride, polyethylene, polyamide,polypropylene, polysulfone, polyurethane, and co-polymers of these andother polymers. Gasket 416 is flat, circumferentially surroundsperforated member 401 and filter 403, and is made from rubber, silicone,nitrile rubber, polytetrafluoroethylene, a plastic polymer such aspolychlorotrifluoroethylene, or other readily compressible material.

Retentate member 404 comprises a generally smooth upper surface; asingle distribution channel 405 (here, located centrally) whichtraverses retentate member 404 from its top surface to its bottomsurface and for most of the length of retentate member 404 (details ofwhich are described in relation to FIG. 4I); and ridges 406 a, whichhere are disposed on the bottom surface of retentate member 404 (e.g.,adjacent perforated member 401) where the ridges 406 a traverse thebottom of retentate member 404 from side-to-side (e.g., left to right)(in this embodiment, there are approximately 28 ridges 406 a). As usedherein with respect to the distribution channels in the retentate memberor permeate member, “most of the length” means about 95% of the lengthof the retentate member or permeate member, or about 90%, 85%, 80%, 75%,or 70% of the length of the retentate member or permeate member. Flowdirectors 406 are formed between ridges 406 a. In addition, retentatemember 404 has a single port 407, which allows for cells to beintroduced into singulation assembly 420 a; and there is also adistribution channel cover 413, which covers the single distributionchannel 405 in retentate member 404. In this embodiment, distributionchannel 405 is approximately 150 mm long and 1 mm wide; retentate memberridges 406 a are approximately 0.5 mm in height and 80 mm in length; andretentate member flow directors 406 are approximately 5 mm across. Thevolume of fluid in the singulation assembly ranges from 5 mL to 100 mL,or from 7.5 mL to 60 mL, or from 10 mL to 40 mL (note this is for a 200Kperforation singulation assembly).

In addition to retentate member 404, also seen in FIG. 4F are fasteners412, a center “sandwich” layer comprising a gasket 416 surrounding aperforated member 401 disposed above a filter or membrane 403 (theindividual components are not seen in this FIG. 4F). The bottom layer ofsingulation assembly 420 a seen in FIG. 4F is formed by a permeatemember 408. The permeate member 408, like retentate member 404,comprises one or more permeate member distribution channels, permeatemember ridges, permeate member flow directors, and one or more ports(none of which are shown in FIG. 4F but see FIG. 4G). The singulationassemblies 420 and SWIIN modules 400 comprising the singulationassemblies 420 are fabricated from material that withstand temperatureof 4° C. to 60° C. Heating and cooling of the SWIIN modules is providedby a Peltier device or thermoelectric cooler; or a combination ofsystems may be employed in, e.g., multi-layer SWIIN modules (see, e.g.,FIGS. 4BB and 4CC) such as reverse Rankine vapor-compressionrefrigeration or absorption heat pumps.

In the solid wall singulation or substantial singulation, growth,induction of editing and normalization and/or cherry-picking modules(“solid wall isolation/induction/normalization module” or “SWIIN”)described in FIGS. 4A-4CC, cells and medium (at a dilution appropriatefor Poisson or substantial Poisson distribution of the cells in themicrowells of the perforated member) are flowed into distributionchannel 405 from one or more ports (in FIG. 4F, there is one port 407)in retentate member 404, and the cells settle in the microwells, again,in a Poisson or substantial Poisson distribution of the cells in themicrowells. The cells are retained in the microwells as they cannottravel through filter 403. After cell loading, an appropriate medium maythen be introduced into the singulation assembly via the retentatemember; that is, medium is introduced through ports 411 (FIG. 4G), andthe medium travels into distribution channels 409 (FIG. 4G) in permeatemember 408, then is distributed throughout flow directors 410 (FIG. 4G)in permeate member 408. The medium in permeate member 408 can flowupward through filter 403 so as to nourish the cells loaded into themicrowells. In operation, once the cells are deposited into themicrowells, they are grown for an initial, e.g., 2-100 or so doublings,editing is induced by, e.g., raising the temperature of the SWIIN to 42°C. to induce a temperature inducible promoter or by removing growthmedium from the permeate member (back through ports 411, see FIG. 4G)and by replacing the growth medium with a medium comprising a chemicalcomponent that induces an inducible promoter. Once editing has takenplace, the temperature or the SWIIN may be decreased, or the inducingmedium may be removed and replaced with fresh medium lacking thechemical component and thus deactivating the inducible promoter. Thecells then are allowed to continue to grow in the SWIIN until the growthof the cell colonies in the microwells is normalized. Once normalizationhas taken place, the colonies are flushed from the microwells (e.g., byapplying pressure to permeate member 408 and thus filter 403) and thecells in the colonies are pooled; or, alternatively, the growth of thecell colonies in the microwells is monitored, and slow-growing coloniesare directly selected by, e.g., pooling the cells from the slow-growingcolonies by pipetting slow-growing cells from the microwells into a tubeor other vessel, or colonies from individuals microwells can be selectedand pipetted into, e.g., individual wells in a 96-well or 384-wellplate.

Colony growth in the singulation assembly (and thus in the SWIIN module)can be monitored by automated devices such as those sold by JoVE(ScanLag™ system, Cambridge, Mass.) (also see Levin-Reisman, et al.,Nature Methods, 7:737-39 (2010)). Cell growth for, e.g., mammalian cellsmay be monitored by, e.g., the growth monitor sold by IncuCyte (AnnArbor, Mich.) (see also, Choudhry, PLos One, 11(2):e0148469 (2016)).Further, automated colony pickers may be employed, such as those soldby, e.g., TECAN (Pickolo™ system, Mannedorf, Switzerland); Hudson Inc.(RapidPick™, Springfield, N.J.); Molecular Devices (QPix 400™ system,San Jose, Calif.); and Singer Instruments (PIXL™ system, Somerset, UK).

FIG. 4G depicts the embodiment of the singulation assembly 420 a in FIG.4F from a bottom perspective view, which presents the bottom of thepermeate member 408 of singulation assembly 420 a. Permeate member 408comprises a generally smooth lower surface (which in FIG. 4G, becausesingulation assembly 420 a is viewed from the bottom, is toward the topof FIG. 4G); two distribution channels (not shown) covered bydistribution channel covers 414 here, located on either side (leftside/right side) of the bottom of permeate member 408. Also seen areridges 410 a, which here are disposed on the top surface of permeatemember 408 (where the top surface of the permeate member 408 in FIG. 4Gis facing down) and traverse the top of permeate member 408 fromright-to-left (in this embodiment, there are approximately 28 ridges 410a), with flow directors 410 formed ridges 410 a. In addition, permeatemember 408 has two ports 411, which deliver fluids to and remover fluidsfrom the distribution channels (not seen here as they are covered bydistribution channel covers 414).

In addition to permeate member 404, also seen in FIG. 4F are fasteners412, a center “sandwich” layer comprising a gasket 416 surrounding aperforated member 401 swaged with a filter or membrane 403 (theindividual components are not seen in this FIG. 4G). The bottom-mostlayer of singulation assembly 420 a seen in FIG. 4G is retentate member404. It should be noted that in the embodiments depicted in FIGS.4F-4CC, fasteners 412 are exemplified; however, other means can be usedto secure retentate member 404, gasket 416, perforated member 401,filter 403, and permeate member 408, such as adhesives—such as apressure sensitive adhesive, ultrasonic welding or bonding, solventbonding, mated fittings, or a combination of adhesives, welding, solventbonding, and mated fittings; and other such fasteners and couplings.

FIG. 4H depicts the embodiment of the singulation assembly 420 a ofFIGS. 4F and 4G from a side exploded perspective view. From the top ofFIG. 4H, is seen retentate member 404 comprising single distributionchannel 405 which traverses retentate member 404 from its top surface toits bottom surface and for most of the length of retentate member 404(details of which are described in relation to FIG. 4I); ridges 406 a,which are disposed on the bottom surface of retentate member 404 andtraverse the bottom surface of retentate member 404 from left-to-right(in this embodiment, there are approximately 26 ridges 406 a), and flowdirectors 406 that are formed between ridges 406 a. In addition,retentate member 404 has a single port 407, which allows for cells andother fluids to be introduced into and removed from retentate member 404and thus into or out of singulation assembly 420 a. There is also adistribution channel cover 413, which is configured to fit into andcover single distribution channel 405.

Gasket 416, perforated member 401 and filter 403 can be seen moreclearly in this exploded view of singulation assembly 420 a, whereperforated member 401 and filter 403 are of very similar size—if not thesame size—and gasket 416 is configured to surround perforated member 401and filter 403 and to provide a leak-proof seal between retentate member404 and permeate member 408. In FIG. 4H, permeate member 408 is seenfrom the top down where permeate member 408 comprises two distributionchannels 409 located lengthwise on either side (left-right) of permeatemember 408, ridges 410 a, which here are disposed on the top surface ofpermeate member 408 and traverse the top of permeate member 408 fromside-to-side (left to right) (in this embodiment, there areapproximately 28 ridges 410 a), and flow directors 410 that are formedbetween ridges 410 a in permeate member 408. In addition, permeatemember 408 has two ports 411 (only one of which is seen in this FIG.4H), where ports 411 deliver fluids and remove fluids from distributionchannels 409. Also seen are distribution channel covers 414 that are notin place covering distribution channels 409. Also seen in FIG. 4H arefasteners 412, although again, other means for securing the componentsof singulation assembly 420 a may be used and preferably are used forproduction singulation assemblies.

FIG. 4I is a close-up side perspective view of the top surface ofretentate member 404, including port 407 (here with a luer connectionfrom a fluid source to port 407 in retentate member 404), distributionchannel 405, which traverses retentate member 404 from its top surfaceto its bottom surface and for most of the length of retentate member 404and is configured to deliver fluids to (and remove fluids from) flowdirectors 406 on the bottom surface of retentate member 408.Distribution channel 405 comprises flow director holes 415 thatcorrespond to flow directors 406, as flow directors 415 are positionedin distribution channel 405 between retentate member ridges 406 a.Distribution channel cover 413 is disposed upon the top surface ofretentate member 404, covering (and forming the top surface of)distribution channel 405.

FIG. 4J is a close-up side cross sectional view of singulation assembly420 a. In FIG. 4J, retentate port 407 is seen (with a luer connectionfrom a fluid source to port 407), as is gasket 416, and perforatedmember 401 and filter 403 swaged together and disposed between retentatemember 404 and permeate member 408. Also seen are ridges 406 a, whichare disposed on the bottom surface of retentate member 404, and flowdirectors 406 that are formed between ridges 406 a. In addition, seenare ridges 410 a permeate member 408, as well as flow directors 410 thatare formed between ridges 410 a. Note that ridges 406 a on retentatemember 404 and ridges 410 a on permeate member 408 meet and arecoincident with one another separated only by perforated member 401 andfilter 403, thus flow directors 406 and flow directors 410 are alsocoincident with one another. Also seen are flow director holes 415 thatcorrespond to flow directors 406, as they are positioned betweenretentate member ridges 406 a. Ridges 406 a on retentate member 404 andridges 410 a on permeate member 408 not only form flow directors 406 and410 in retentate member 404 and permeate member 408, respectively, butprovide support to the singulation assembly 420 by distributing pressurethroughout the singulation assembly 420 and preventing tears in filteror membrane 403.

FIG. 4K depicts another embodiment of a singulation assembly 420 b froma top perspective view, which presents “retentate side” of singulationassembly 420 b. The singulation assembly 420 b embodiment seen in FIG.4K differs from singulation assembly 420 a seen in FIGS. 4F-4H in thatthe retentate member 404 in FIGS. 4K-4M has two distribution channels405 disposed lengthwise on either side (left-right) of retentate member404 instead of a single distribution channel 405 disposed lengthwisedown the middle of retentate member 404. As in FIGS. 4F-4H, retentatemember 404 of FIGS. 4J-4M comprises a generally smooth upper surface;two distribution channels 405 which traverse retentate member 404 fromits top surface to its bottom surface and for most of the length ofretentate member 404; and ridges 406 a, which are disposed on the bottomsurface of retentate member 404 and traverse the bottom of retentatemember 404 from side-to-side, left-to-right (in this embodiment, thereare approximately 28 retentate member ridges 406 a). Flow directors 406are formed between ridges 406 a on retentate member 404. In addition,retentate member 404 has two ports 407, which allow for cells and mediumto be introduced into (and removed from) singulation assembly 420 b.There are also distribution channel covers 413, which cover the twodistribution channels 405 and actually provide the top surface ofdistribution channels 405.

In addition to retentate member 404, also seen in FIG. 4K are fasteners412, the center “sandwich” layer comprising a gasket 416 surroundingperforated member 401 swaged with (and positioned above) filter ormembrane 403 (the individual components are not seen in this FIG. 4K).The bottom layer of singulation assembly 420 b seen in FIG. 4K is formedby a permeate member 408. Permeate member 408, like retentate member404, comprises one or more (as seen in FIG. 4L, there are two) permeatemember distribution channels, a multiplicity of ridges, and flowdirectors, and one or more ports (none of which are shown in FIG. 4K butsee FIG. 4L).

As described before in relation to FIGS. 4F-4H, in the solid wallsingulation or substantial singulation, growth, induction of editing andeither normalization or cherry picking modules (“solid wallisolation/induction/normalization module” or “SWIIN”) described in FIGS.4F-4CC, cells and medium (at a dilution appropriate for Poisson orsubstantial Poisson distribution of the cells in the microwells of theperforated member) are flowed into distribution channels 405 from thetwo ports 407 in retentate member 404, and the cells settle in themicrowells, again, in a Poisson or substantial Poisson distribution ofthe cells in the microwells. The cells are retained in the microwells ofperforated member 401 as the cells cannot travel through filter 403. Anappropriate medium is introduced into singulation assembly 420 b throughpermeate member 408. The medium flows upward through filter 403 tonourish the cells. Thus, in operation, the cells are deposited into themicrowells, are grown for an initial, e.g., 2-100 doublings, editing isinduced by, e.g., raising the temperature of the SWIIN to 42° C. toinduce a temperature inducible promoter or by removing growth mediumfrom the permeate member and replacing the growth medium with a mediumcomprising a chemical component that induces an inducible promoter. Onceediting has been allowed to take place, the temperature of the SWIIN maybe decreased, or the inducing medium may be removed and replaced withfresh medium lacking the chemical component thereby activating theinducible promoter. The cells then are allowed to continue to grow inthe SWIIN until the growth of the cell colonies in the microwells isnormalized. Once the colonies are normalized, the colonies are flushedfrom the microwells (by applying pressure to the permeate memberchannels and flow directors and thus to the filter) and pooled;alternatively, the growth of the cell colonies in the microwells ismonitored, and slow-growing colonies are directly selected by, e.g.,pooling the cells from the slow-growing colonies.

FIG. 4L depicts the embodiment of the singulation assembly 420 b in FIG.4K from a bottom perspective view, which presents the bottom of thepermeate member 408 of singulation assembly 420 b. Permeate member 408comprises a generally smooth lower surface (which in FIG. 4L, becausesingulation assembly 420 b is viewed from the bottom, is toward the topof FIG. 4L); two distribution channels (not shown) covered bydistribution channel covers 414 (here, located on either side(left-right) of permeate member 408), ridges 410 a, which are disposedon the top surface of permeate member 408 (where the top surface of thepermeate member 408 in FIG. 4L is facing down) and traverse the top ofpermeate member 408 from side-to-side (in this embodiment, there areapproximately 28 ridges 410 a), and flow directors 410 that are formedbetween ridges 410 a. In addition, permeate member 408 has two ports411, which deliver fluids to (and remove fluids from) the distributionchannels (not seen here as they are covered by distribution channelcovers 414) and then to flow directors 410.

In addition to permeate member 404, also seen in FIG. 4L are fasteners412, a center “sandwich” layer comprising a gasket 416 surrounding aperforated member 401 swaged with and positioned above a filter ormembrane 403 (the individual components are not seen in this FIG. 4L),and the bottom-most layer of singulation assembly 420 b seen in FIG. 4Lis retentate member 404. Again, it should be noted that in theembodiments depicted in FIGS. 4F-4CC, fasteners are exemplified;however, other means can be used to secure retentate member 408, gasket416, perforated member 401, filter 403, and permeate member 408, such asadhesives, ultrasonic welding or bonding, solvent bonding, matedfittings, or a combination of adhesives, welding, solvent bonding, andmated fittings; and other such fasteners and couplings.

FIG. 4M depicts the embodiment of the singulation assembly 420 b ofFIGS. 4K and 4L from a side exploded perspective view. From the top ofFIG. 4M is seen retentate member 404 comprising two distributionchannels 405 located on either side (left-right) of retentate member404, both of which traverse retentate member 404 from its top surface toits bottom surface and for most of the length of retentate member 404;ridges 406 a, which are disposed on the bottom surface of retentatemember 404 and traverse the bottom surface of retentate member 404 fromside-to-side (in this embodiment, there are approximately 28 ridges 406a), and flow directors 406 that are formed between ridges 406 a. Inaddition, retentate member 404 has two ports 407, which allow for cellsand other fluids to be introduced into (and removed from) singulationassembly 420 a through distribution channels 405. There is also twodistribution channel covers 413, which are configured to fit into andcover the two distribution channels 405.

Gasket 416, perforated member 401 and filter 403 can be seen moreclearly in this exploded view of singulation assembly 420 b, whereperforated member 401 and filter 403 are of very similar size and gasket416 is configured to surround perforated member 401 and filter 403, tosecure perforated member 401 and filter 403, and to provide a leak-proofseal between retentate member 404 and permeate member 408. In FIG. 4M,permeate member 408 is seen from the top down where permeate member 408comprises two distribution channels 409 located lengthwise on eitherside (left-right) of permeate member 408, both of which traversepermeate member 408 from its bottom surface to its top surface and formost of the length of permeate member 408, ridges 410 a, which here aredisposed on the top surface of permeate member 408 and traverse the topof permeate member 408 from side-to-side, and flow directors 410 thatare formed between ridges 410 a. In addition, permeate member 408 hastwo ports 411, seen in this FIG. 4M as ports on retentate member 404,which when singulation assembly 420 b is assembled are fluidicallyconnected to distribution channels 409 on permeate member 408, which areconnected to flow distributors 410, and thus to filter 403 disposed uponpermeate member 408. Also seen are distribution channel covers 414 thatare separate from singulation assembly 420 b in this exploded view andnot inserted into permeate member 408 to cover distribution channels409. Also seen in FIG. 4M are holes for fasteners 412 (fasteners notshown), although again, other means aside from fasteners may be used forsecuring the components of singulation assembly 420 b.

FIG. 4N depicts another embodiment of a singulation assembly 420 c froma top perspective view, which presents the “retentate side” ofsingulation assembly 420 c. The singulation assembly 420 c embodimentseen in FIG. 4N differs from singulation assembly 420 a seen in FIGS.4F-4H in that the retentate member 404 in FIGS. 4N-4P has twodistribution channels 405 disposed lengthwise on either side(left-right) of retentate member 404 instead of a single distributionchannel 405 disposed lengthwise down the middle of retentate member 404.The singulation assembly 420 c embodiment seen in FIG. 4N differs fromsingulation assembly 420 b seen in FIGS. 4K-4M in that the configurationof the distribution channels 405 in retentate member 404 (anddistribution channels 409 in permeate member 408) in FIGS. 4N-4P isdifferent from that in FIGS. 4K-4M. The distribution channels 405 (anddistribution channels 409) in FIGS. 4N-4P are branched; that is, insteadof a port 407 delivering fluids directly into a distribution channel 405at the point where port 407 intersects directly with distributionchannel 405, in the embodiment of the singulation assembly shown inFIGS. 4N-4P, fluids flow into ports 407, then into branched distributionchannels 405 on either side (left-right) of retentate member 407. Thebranched distribution channels have a first conduit which terminatesapproximately half-way down the length of retentate member 404, wherethe first conduit then branches into two secondary conduits, the twosecondary conduits branch into four tertiary conduits, and thesetertiary conduits deliver fluids to a final conduit that travels thelength of the retentate member, evenly distributing delivering fluids tothe flow directors 406.

Note that any configuration of distribution channels (e.g., distributionchannels 405 on retentate member 404 and distribution channels 409 onpermeate member 408) may be used on retentate member 404 or permeatemember 408, as long as the distribution channels adequately distributefluids to flow directors 406 or flow directors 410. As seen in FIGS.4F-4H, the configuration of distribution channels 405 on retentatemember 404 and distribution channels 409 on permeate member 408 may bedifferent, or as seen in FIGS. 4K-4M and FIGS. 4N-4P, the configurationof distribution channels 405 on retentate member 404 and permeate memberdistribution channels 409 on permeate member 408 may be the same. As inFIGS. 4K-4M, retentate member 404 of FIGS. 4N-4P comprises a generallysmooth upper surface; two distribution channels 405 one of the left andone on the right of retentate member 404 which traverse retentate member404 from its top surface to its bottom surface and are branched thusdistributing fluid the length of retentate member 404.

Retentate member 404 also comprises ridges 406 a, which are disposed onthe bottom surface of retentate member 404 and traverse the bottom ofretentate member 404 from side-to-side. In addition—and as in previousembodiments—there are flow directors 406 that are formed between ridges406 a on retentate member 404; and two ports 407, which introduce anddistribute cells and medium into (and remove cells and medium from)retentate member 404 of singulation assembly 420 c. Also seen aredistribution channel covers 413, which cover the two brancheddistribution channels 405 on retentate member 404 and actually providethe top surface of branched distribution channels 405. Note that thebranching for distribution channels 405 is a part of retentate member404 in this embodiment; however, retentate member 404 may not comprisethe branches and the branching may be featured on distribution channelcovers 413, which are mated to retentate member 404.

In addition to retentate member 404, also seen in FIG. 4N are fasteners412, the center “sandwich” layer comprising a gasket 416 surroundingperforated member 401 swaged with and positioned above filter ormembrane 403. The bottom layer of singulation assembly 420 c seen inFIG. 4N is formed by permeate member 408. Permeate member 408, likeretentate member 404, comprises one or more (as seen in FIG. 4O, thereare two) distribution channels (here, they are branched), ridges, flowdirectors, and one or more ports (none of which are shown in FIG. 4N butsee FIG. 4O).

FIG. 4O depicts the embodiment of the singulation assembly 420 c in FIG.4N from a bottom perspective view, which presents the bottom of thepermeate member 408 of singulation assembly 420 c. Permeate member 408comprises a generally smooth lower surface (which in FIG. 4O, becausesingulation assembly 420 c is viewed from the bottom, is toward the topof FIG. 4O); two branched distribution channels 409 covered bydistribution channel covers 414, ridges 410 a, disposed on the topsurface of permeate member 408 (where the top surface of the permeatemember 408 in FIG. 4O is facing down) and traverse the top surface ofpermeate member 408 from side-to-side, and flow directors 410 that areformed between ridges 410 a. In addition, permeate member 408 has twoports 411, which deliver fluids to the branched distribution channels409 and to flow directors 410. As with retentate member 404 anddistribution channel covers 413, the branching for distribution channels409 in permeate member 408 is a part of permeate member 408; however,permeate member 408 may not comprise the branches and the branching mayinstead be featured on distribution channel covers 414, which are matedto permeate member 408.

In addition to permeate member 404, also seen in FIG. 4O are fasteners412, a center “sandwich” layer comprising a gasket 416 surrounding aperforated member 401 disposed above a filter or membrane 403 (theindividual components are not seen in this FIG. 4M), and bottom-mostlayer of singulation assembly 420 a seen in FIG. 4O is the retentatemember 404.

FIG. 4P depicts the embodiment of the singulation assembly 420 c ofFIGS. 4N and 4O from a side exploded perspective view. From the top ofFIG. 4P is seen retentate member 404 comprising two brancheddistribution channels 405 located on either side (left-right) ofretentate member 404, both of which traverse retentate member 404 fromits top surface to its bottom surface where the branches traverse mostof the length of retentate member 404; ridges 406 a, which are disposedon the bottom surface of retentate member 404 and traverse the bottomsurface of retentate member 404 from side-to-side, and flow directors406 that are formed between ridges 406 a. In addition, retentate member404 has two ports 407, which are fluidically coupled to brancheddistribution channels 405 and flow directors 406 and are configured tointroduce cells and other fluids into (and remover cells and otherfluids from) singulation assembly 420 a. There are also distributionchannel covers 413, which are configured to fit into and cover the twobranched distribution channels 405.

Gasket 416, perforated member 401 and filter 403 can be seen clearly inthis exploded view of singulation assembly 420 c, where perforatedmember 401 and filter 403 are of very similar size and gasket 416 isconfigured to surround perforated member 401 and filter 403 and toprovide a leak-proof seal between retentate member 404 and permeatemember 408. In FIG. 4P, permeate member 408 is seen from the top downwhere permeate member 408 comprises two branched distribution channels409 located lengthwise on either side (left-right) of permeate member408, both of which traverse permeate member 408 from its bottom surfaceto its top surface and provide branched conduits for most of the lengthof permeate member 408. Permeate member 408 further comprises ridges 410a, which here are disposed on the top surface of permeate member 408 andtraverse the top of permeate member 408 from side-to-side and flowdirectors 410 that are formed between permeate member ridges 410 a. Inaddition, permeate member 408 has two ports 411 (only one port 411 canbe seen in FIG. 4P). Also seen are distribution channel covers 414 thatare separate from singulation assembly 420 c in this exploded view andnot inserted to cover branched distribution channels 409. Also seen inFIG. 4N are fasteners 412, although again, other means aside fromfasteners may be used for securing the components of singulationassembly 420 c.

FIG. 4Q is a close-up top view from the top of retentate member 404 asshown in FIGS. 4N and 4P. Seen are ridges 406 a, which are disposed onthe bottom surface of retentate member 404 and traverse the bottomsurface of retentate member 404 from side-to-side, and flow directors406 that are formed between ridges 406 a. Additionally seen isdistribution channel 405 comprising flow director holes configured todistribute fluid into flow directors 406, where distribution channel 405is covered by distribution channel cover 413.

FIG. 4R is a close-up cross-sectional view of retentate member 404, withridges 406 a and flow directors 406 that are formed between ridges 406a. Also seen is permeate member 408, with ridges 410 a and flowdirectors 410 that are formed between permeate member ridges 410 a.Interposed between retentate member 404 and permeate member 408 aregasket 416, perforated member 401, and filter 403. Note that ridges 406a on retentate member 404 and ridges 410 a are coincident with oneanother, separated only by perforated member 401 and filter 403. Asstated previously, ridges 406 a and ridges 410 a provide support toperforated member 401 and filter 403 and reduce the likelihood thatfilter 403 will tear during, e.g., cell loading or medium exchange.

FIGS. 4S-4W depict different views of one embodiment of a solid wallsingulation or substantial singulation, growth, induction of editing andeither normalization and cherry picking (SWIIN) module 400. FIG. 4Spresents a side perspective view of SWIIN module 400. SWIIN module 400comprises, e.g., one of the exemplary singulation assemblies seen inFIGS. 4F-4H, 4K-4M and 4N-4P which are housed in a SWIIN cover 440 andare one part of the SWIIN module. Various components of SWIIN cover 440include reservoir cover 442, grip 441, windows 444 (shown are sixwindows, 444 a, 444 b, 444 c, 444 d, 444 e, 444 f), feet 443, andbarcode (or other identifying information) 445. Note that in theembodiments depicted in FIGS. 4S-4Z the windows are round; however, itshould be recognized by one of ordinary skill in the art that thewindows can be any shape. Typically, it is desirable for 30% or more ofthe retentate member be available for viewing to get a reasonablesub-sample statistic on cell loading. Also seen is reservoir assemblycover 430 a, which in this embodiment is not molded with SWIIN cover 440but resides within the reservoir cover portion 442 of SWIIN cover 440.Reservoir assembly cover 430 a comprises four reservoir access apertures(432 a, 432 b, 432 c, and 432 d) and four pneumatic access apertures(433 a, 433 b, 433 c, and 433 d). Pneumatic access apertures 433 a, 433b, 433 c, and 433 d in most embodiments include filters to preventcontamination.

Windows 444 may be used to monitor cell loading and/or cell growth viacamera (e.g., video camera). For example, a video camera may be used tomonitor cell growth by, e.g., density change measurements based on animage of an empty well, with phase contrast, or if, e.g., a chromogenicmarker, such as a chromogenic protein, is used to add a distinguishablecolor to the cells. Chromogenic markers such as blitzen blue, dreidelteal, virginia violet, vixen purple, prancer purple, tinsel purple,maccabee purple, donner magenta, cupid pink, seraphina pink, scroogeorange, and leor orange (the Chromogenic Protein Paintbox, all availablefrom ATUM (Newark, Calif.)) obviate the need to use fluorescence,although fluorescent cell markers, fluorescent proteins, andchemiluminescent cell markers may also be used.

FIG. 4T is a top-down view of SWIIN module 400, showing SWIIN cover 440and various components thereof including reservoir cover 442, grip 441,windows 444 (shown are six windows), feet 443, and barcode (or otheridentifying information) 445. Also seen is reservoir assembly cover 430a residing within the reservoir cover portion 442 of SWIIN cover 440,where reservoir assembly cover 430 a comprises four reservoir accessapertures (432 a, 432 b, 432 c, and 432 d) and four pneumatic accessapertures (433 a, 433 b, 433 c, and 433 d).

FIG. 4U is a side view of SWIIN module 400, depicting SWIIN cover 440,reservoir cover 442, grip 441, and feet 443. FIG. 4V is a view from thebarcode “end” of SWIIN module 400 toward the reservoir “end” of SWIINmodule 400. Seen are SWIIN cover 400, reservoir cover 442, grip 441, andfeet 443. FIG. 4W is a bottom perspective view of SWIIN module 400,showing SWIIN cover 440, reservoir cover 442, feet 443, as well asdistribution channel covers 414 disposed on the bottom of permeatemember 408.

FIGS. 4X-4Z depict views of a reservoir assembly 430. In FIG. 4X,reservoir assembly 430 is disposed upon a singulation assembly 420comprising retentate member 404; distribution channel covers 413; agasket, perforated member, and filter assembly (not shown clearly), anda permeate member (also not shown clearly) Reservoir assembly 430comprises reservoir assembly cover 430 a, where reservoir assembly cover430 a comprises four reservoir access apertures (432 a, 432 b, 432 c,and 432 d) and four pneumatic access apertures (433 a, 433 b, 433 c, and433 d).

FIG. 4Y is a top perspective view of a cross section of reservoirassembly 430 taken through the “reservoir” end of SWIIN cover 440. Seenare feet 443 of SWIIN cover 440. Reservoirs 431 a, 431 b, 431 c, and 431d are seen, as well as reservoir/channel ports 434 a, 434 b, 434 c, and434 d. Reservoirs 431 b and 431 c are fluidically coupled to conduitsthat either are fluidically coupled to distribution channels on theretentate member or distribution channels on the permeate member, andreservoirs 431 a and 431 d are fluidically coupled to conduits thateither are fluidically coupled to distribution channels on the retentatemember or distribution channels on the permeate member; that is ifreservoirs 431 b and 431 c are fluidically coupled to conduits that arecoupled to distribution channels on the retentate member, thenreservoirs 431 a and 431 d are fluidically coupled to conduits that arecoupled to distribution channels on the permeate member. However, anyreservoir may be configured to deliver fluids to and remove fluids fromeither retentate member 404 or permeate member 408. Reservoirs 431 a,431 b, 431 c, and 431 d typically have a volume of from 5.0 to 100 mL,or from 7.5 to 60 mL, or from 10 to 40 mL (these volumes are for the200K singulation assembly). Note that reservoirs 431 a, 431 b, 431 c,and 431 d are funnel-shaped in the portion of the reservoir leading toreservoir/channel ports 434 a, 434 b, 434 c, and 434 d and aid indelivery of fluids from reservoirs 431 a, 431 b, 431 c, and 431 d.

FIG. 4Z shows three different views of four co-joined reservoirs 435.The top figure is a top-down view of co-joined reservoirs 435, themiddle figure is a side view of co-joined reservoirs 435, and the bottomfigure is a bottom-up view of co-joined reservoirs 435. Reservoirs 431a, 431 b, 431 c, and 431 d are seen in all figures, andreservoir/channel ports 434 a, 434 b, 434 c, and 434 d are seen in thetop and bottom figures.

FIG. 4AA is an exemplary pneumatic block diagram suitable for the SWIINmodule depicted in FIGS. 44S-4W and, e.g., utilizing the singulationassemblies described in relation to FIGS. 4F-4R. Note that there are twosolenoid valves (SV) for each reservoir-two retentate reservoirs (RR1and RR2) and two permeate reservoirs (PR1 and PR2)—where one of thevalves is for pneumatic actuation, and one of the valves serves to blockthe line. Note there is a flow meter for each pair of solenoid valves.Also, in this embodiment there is a single manifold serving allreservoirs; however, other embodiments may employ two manifolds, withPR1 and RR1 served by one manifold and PR2 and RR2 served by anothermanifold, or each reservoir (PR1, RR1, PR2, RR2) served by a separatemanifold.

FIG. 4BB shows a double-layer SWIIN module 460, with a housing thatcomprises two singulation assemblies (singulation assemblies are notseen in this FIG. 4AA). FIG. 4CC graphically depicts a quadruple layerSWIIN module 465 comprising four singulation assemblies 420. FIG. 4BBgraphically depicts a peltier device 466 and a fan 467 with forcedairflow 468 that can be used to keep the four singulation assemblies 420at a desired temperature; however, note that in multi-layer SWIINs, acombination of systems may be used such as reverse-Rankinevapor-compression refrigeration, or absorption heat pumps.

FIG. 4DD is an exemplary pneumatic architecture diagram for a doublelayer SWIIN showing, along with Tables 5 and 6, one embodiment ofpneumatics employed to singulate or substantially singulate, grow,initiate editing, and normalize cells in the SWIIN module described inrelation to FIG. 4BB. Looking at FIG. 4DD, four permeate reservoirs areseen (PR1, PR2, PR3, and PR4) and four retentate reservoirs are seen(RR1, RR2, RR3 and RR4). There are four flow meters (FM1, FM2, FM3 andFM4), ten 3-way solenoid valves (“3-way SV”), a pressure sensor, a propvalve to adjust pressure, and a pump capable of delivering pressures of−5 to 5 psi. The designation NC is for “normally closed”, NO is for“normally open”, and C is “closed”. Table 5 provides, for each step ofthe cell concentration process, the status of each valve shown in FIG.4CC and the pressure detected by pressure sensors 1 and 2. In Table 5,for the pump, 1=on, and 0=off. For the solenoid valves, 1=energized, and0=de-energized. Table 6 provides, for each step of the cellconcentration process, the volume in mL of fluid in each reservoir(i.e., the four retentate reservoirs and the four permeate reservoirs).Perforated member 401 is shown, as is temperature zone 499.

In addition to the solid wall cell singulation device (SWIIN) describedin relation to FIGS. 3A-3E and 4A-4BB, other cell singulation (orsubstantial singulation) devices may be employed in the multi-modulecell processing instrument, such as those described in U.S. Ser. No.62/735,365, entitled “Detection of Nuclease Edited Sequences inAutomated Modules and Systems”, filed 24 Sep. 2018, and U.S. Ser. No.62/781,112, entitled “Improved Detection of Nuclease Edited Sequences inAutomated Modules and Systems”, filed 18 Dec. 2018, includingsingulation or substantial singulation by plating on agar, singulationor substantial singulation by isolating cells on functionalized islands,singulation or substantial singulation within aqueous droplets carriedin a hydrophobic carrier fluid or Gel Beads-in-Emulsion (GEMs, see,e.g., 10× Genomics, Pleasanton, Calif.), or singulation or substantialsingulation within a polymerized alginate scaffold (for this embodimentof singulation, also see U.S. Ser. No. 62/769,805, entitled “ImprovedDetection of Nuclease Edited Sequences in Automated Modules andInstruments via Bulk Cell Culture”, filed 20 Nov. 2018).

Automated Cell Editing Instruments and Modules

Automated Cell Editing Instruments

FIG. 5A depicts an exemplary automated multi-module cell processinginstrument 500 to, e.g., perform one of the exemplary workflowsdescribed infra, comprising one or more reagent cartridges as describedherein. The instrument 500, for example, may be and preferably isdesigned as a stand-alone desktop instrument for use within a laboratoryenvironment. The instrument 500 may incorporate a mixture of reusableand disposable components for performing the various integratedprocesses in conducting automated genome cleavage and/or editing incells. Illustrated is a gantry 502, providing an automated mechanicalmotion system (actuator) (not shown) that supplies XYZ axis motioncontrol to, e.g., an automated (i.e., robotic) liquid handling system558 including, e.g., an air displacement pipettor 532 which allows forcell processing among multiple modules without human intervention. Insome automated multi-module cell processing instruments, the airdisplacement pipettor 532 is moved by gantry 502 and the various modulesand reagent cartridges remain stationary; however, in other embodiments,the liquid handling system 558 may stay stationary while the variousmodules and reagent cartridges are moved. Also included in the automatedmulti-module cell processing instrument 500 is reagent cartridge 510comprising reservoirs 512 and transformation module 530 (e.g., aflow-through electroporation device as described in detail in relationto FIGS. 8A-8E), as well as a wash cartridge 504 comprising reservoirs506. The wash cartridge 504 may be configured to accommodate largetubes, for example, wash solutions, or solutions that are used oftenthroughout an iterative process. In one example, wash cartridge 504 maybe configured to remain in place when two or more reagent cartridges 510are sequentially used and replaced. Although reagent cartridge 510 andwash cartridge 504 are shown in FIG. 5A as separate cartridges, thecontents of wash cartridge 504 may be incorporated into reagentcartridge 510. The reagent cartridge 510 and wash cartridge 504 may beidentical except for the consumables (reagents or other componentscontained within the various inserts) inserted therein. Note in thisembodiment transformation module 530 is contained within reagentcartridge 510; however, in alternative embodiments transformation module530 is contained within its own module or may be part of another module,such as a growth module.

In some implementations, the wash and reagent cartridges 504 and 510comprise disposable kits (one or more of the various inserts andreagents) provided for use in the automated multi-module cellprocessing/editing instrument 500. For example, a user may open andposition each of the reagent cartridge 510 and the wash cartridge 504comprising various desired inserts and reagents within a chassis of theautomated multi-module cell editing instrument 500 prior to activatingcell processing.

Also illustrated in FIG. 5A is the robotic liquid handling system 558including the gantry 502 and air displacement pipettor 532. In someexamples, the robotic handling system 558 may include an automatedliquid handling system such as those manufactured by Tecan Group Ltd. ofMannedorf, Switzerland, Hamilton Company of Reno, Nev. (see, e.g.,WO2018015544A1), or Beckman Coulter, Inc. of Fort Collins, Colo. (see,e.g., US20160018427A1). Pipette tips may be provided in a pipettetransfer tip supply (not shown) for use with the air displacementpipettor 532.

Inserts or components of the wash and reagent cartridges 504, 510, insome implementations, are marked with machine-readable indicia (notshown), such as bar codes, for recognition by the robotic handlingsystem 558. For example, the robotic liquid handling system 558 may scanone or more inserts within each of the wash and reagent cartridges 504,510 to confirm contents. In other implementations, machine-readableindicia may be marked upon each wash and reagent cartridge 504, 510, anda processing system (not shown, but see element 526 of FIG. 5B) of theautomated multi-module cell editing instrument 500 may identify a storedmaterials map based upon the machine-readable indicia. The exemplaryautomated multi-module cell processing instrument 500 of FIG. 5A furthercomprises a cell growth module 534. (Note, all modules recited brieflyhere are described in greater detail below.) In the embodimentillustrated in FIG. 5A, the cell growth module 534 comprises two cellgrowth vials 518, 520 (described in greater detail below in relation toFIGS. 6A-6D) as well as a cell concentration module 522 (described indetail in relation to FIGS. 7A-7H). In alternative embodiments, the cellconcentration module 522 may be separate from cell growth module 534,e.g., in a separate, dedicated module. Also illustrated as part of theautomated multi-module cell processing instrument 500 of FIG. 5A is asingulation module 540, served by, e.g., robotic liquid handing system558 and air displacement pipettor 532. Also seen are an optional nucleicacid assembly/desalting module 514 comprising a reaction chamber or tubereceptacle (not shown) and a magnet 516 to allow for purification ofnucleic acids using, e.g., magnetic solid phase reversibleimmobilization (SPRI) beads (Applied Biological Materials Inc.,Richmond, BC. The cell growth module, cell concentration module,transformation module, reagent cartridge, and nucleic acid assemblymodule are described in greater detail infra, and an exemplarysingulation module is described in detail in relation to FIGS. 3A-3E and4A-4BB supra.

FIG. 5B is a plan view of the front of the exemplary multi-module cellprocessing instrument 500 depicted in FIG. 5A. Cartridge-based sourcematerials (such as in reagent cartridge 510), for example, may bepositioned in designated areas on a deck 502 of the instrument 500 foraccess by a robotic handling instrument (not shown in this figure). Asillustrated in FIG. 5B, the deck may include a protection sink such thatcontaminants spilling, dripping, or overflowing from any of the modulesof the instrument 500 are contained within a lip of the protection sink.In addition to reagent cartridge 510, also seen in FIG. 5B is washcartridge 504, singulation module 540, and a portion of growth module534. Also seen in this view is touch screen display 550, transformationmodule controls 538, electronics rack 536, and processing system 526.

FIGS. 5C through 5D illustrate side and front views, respectively, ofmulti-module cell processing instrument 500 comprising chassis 590 foruse in desktop versions of the automated multi-module cell editinginstrument 500. For example, the chassis 590 may have a width of about24-48 inches, a height of about 24-48 inches and a depth of about 24-48inches. Chassis 590 may be and preferably is designed to hold allmodules and disposable supplies used in automated cell processing and toperform all processes required without human intervention (that is,chassis 590 is configured to provide an integrated, stand-aloneautomated multi-module cell processing instrument). Chassis 590 maymount a robotic liquid handling system 558 for moving materials betweenmodules. As illustrated in FIG. 5C, the chassis 590 includes a cover 552having a handle 554 and hinge 556 a (hinges 556 b and 556 c are seen inFIG. 5D) for lifting the cover 552 and accessing the interior of thechassis 590. A cooling grate 564 (FIG. 5C) allows for air flow via aninternal fan (not shown). Further, the chassis 590 is lifted byadjustable feet 570 a, 570 c (feet 570, 570 b are shown in FIG. 5D).Adjustable feet 570 a-570 c, for example, may provide additional airflow beneath the chassis 590. A control button 566, in some embodiments,allows for single-button automated start and/or stop of cell processingwithin the automated multi-module cell processing instrument 500.

Inside the chassis 590, in some implementations, a robotic liquidhandling system 558 is disposed along a gantry 502 above wash cartridge504 (reagent cartridge 510 is not seen in these figures). Controlcircuitry, liquid handling tubes, air pump controls, valves, thermalunits (e.g., heating and cooling units) and other control mechanisms, insome embodiments, are disposed below a deck of the chassis 590, in acontrol box region 568. Also seen in both FIGS. 5C and 5D is singulationdevice or module 540. Nucleic acid assembly module 514 comprising amagnet 516 is seen in FIG. 5D.

Although not illustrated, in some embodiments a display screen may bepositioned on the front face of the chassis 590, for example covering aportion of the cover (e.g., see display 550 in FIG. 5B). The displayscreen may provide information to the user regarding the processingstatus of the automated multi-module cell editing instrument 500. Inanother example, the display screen may accept inputs from the user forconducting the cell processing.

The Rotating Growth Module

FIG. 6A shows one embodiment of a rotating growth vial 600 for use withthe cell growth device described herein. The rotating growth vial 600 isan optically-transparent container having an open end 604 for receivingliquid media and cells, a central vial region 606 that defines theprimary container for growing cells, a tapered-to-constricted region 618defining at least one light path 610, a closed end 616, and a driveengagement mechanism 612. The rotating growth vial 600 has a centrallongitudinal axis 620 around which the vial rotates, and the light path610 is generally perpendicular to the longitudinal axis of the vial. Thefirst light path 610 is positioned in the lower constricted portion ofthe tapered-to-constricted region 618. Optionally, some embodiments ofthe rotating growth vial 600 have a second light path 608 in the taperedregion of the tapered-to-constricted region 618. Both light paths inthis embodiment are positioned in a region of the rotating growth vialthat is constantly filled with the cell culture (cells+growth media) andare not affected by the rotational speed of the growth vial. The firstlight path 610 is shorter than the second light path 608 allowing forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a high level (e.g., later in the cell growthprocess), whereas the second light path 608 allows for sensitivemeasurement of OD values when the OD values of the cell culture in thevial are at a lower level (e.g., earlier in the cell growth process).

The drive engagement mechanism 612 engages with a motor (not shown) torotate the vial. In some embodiments, the motor drives the driveengagement mechanism 612 such that the rotating growth vial 600 isrotated in one direction only, and in other embodiments, the rotatinggrowth vial 600 is rotated in a first direction for a first amount oftime or periodicity, rotated in a second direction (i.e., the oppositedirection) for a second amount of time or periodicity, and this processmay be repeated so that the rotating growth vial 600 (and the cellculture contents) are subjected to an oscillating motion. Further, thechoice of whether the culture is subjected to oscillation and theperiodicity therefor may be selected by the user. The first amount oftime and the second amount of time may be the same or may be different.The amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1,2, 3, 4 or more minutes. In another embodiment, in an early stage ofcell growth the rotating growth vial 600 may be oscillated at a firstperiodicity (e.g., every 60 seconds), and then a later stage of cellgrowth the rotating growth vial 600 may be oscillated at a secondperiodicity (e.g., every one second) different from the firstperiodicity.

The rotating growth vial 600 may be reusable or, preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial is consumable and is presented to the user pre-filled withgrowth medium, where the vial is hermetically sealed at the open end 604with a foil seal. A medium-filled rotating growth vial packaged in sucha manner may be part of a kit for use with a stand-alone cell growthdevice or with a cell growth module that is part of an automatedmulti-module cell processing system. To introduce cells into the vial, auser need only pipette up a desired volume of cells and use the pipettetip to punch through the foil seal of the vial. Open end 604 mayoptionally include an extended lip 602 to overlap and engage with thecell growth device. In automated systems, the rotating growth vial 600may be tagged with a barcode or other identifying means that can be readby a scanner or camera (not shown) that is part of the automated system.

The volume of the rotating growth vial 600 and the volume of the cellculture (including growth medium) may vary greatly, but the volume ofthe rotating growth vial 600 must be large enough to generate aspecified total number of cells. In practice, the volume of the rotatinggrowth vial 600 may range from 1-250 mL, 2-100 mL, from 5-80 mL, 10-50mL, or from 12-35 mL. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration andmixing in the rotating growth vial 600. Proper aeration promotes uniformcellular respiration within the growth media. Thus, the volume of thecell culture should be approximately 5-85% of the volume of the growthvial or from 20-60% of the volume of the growth vial. For example, for a30 mL growth vial, the volume of the cell culture would be from about1.5 mL to about 26 mL, or from 6 mL to about 18 mL.

The rotating growth vial 600 preferably is fabricated from abio-compatible optically transparent material—or at least the portion ofthe vial comprising the light path(s) is transparent. Additionally,material from which the rotating growth vial is fabricated should beable to be cooled to about 4° C. or lower and heated to about 55° C. orhigher to accommodate both temperature-based cell assays and long-termstorage at low temperatures. Further, the material that is used tofabricate the vial must be able to withstand temperatures up to 55° C.without deformation while spinning. Suitable materials include cyclicolefin copolymer (COC), glass, polyvinyl chloride, polyethylene,polyamide, polypropylene, polycarbonate, poly(methyl methacrylate(PMMA), polysulfone, polyurethane, and co-polymers of these and otherpolymers. Preferred materials include polypropylene, polycarbonate, orpolystyrene. In some embodiments, the rotating growth vial isinexpensively fabricated by, e.g., injection molding or extrusion.

FIG. 6B is a perspective view of one embodiment of a cell growth device630. FIG. 6C depicts a cut-away view of the cell growth device 630 fromFIG. 6B. In both figures, the rotating growth vial 600 is seenpositioned inside a main housing 636 with the extended lip 602 of therotating growth vial 600 extending above the main housing 636.Additionally, end housings 652, a lower housing 632 and flanges 634 areindicated in both figures. Flanges 634 are used to attach the cellgrowth device 630 to heating/cooling means or other structure (notshown). FIG. 6C depicts additional detail. In FIG. 6C, upper bearing 642and lower bearing 640 are shown positioned within main housing 636.Upper bearing 642 and lower bearing 640 support the vertical load ofrotating growth vial 600. Lower housing 632 contains the drive motor638. The cell growth device 630 of FIG. 6C comprises two light paths: aprimary light path 644, and a secondary light path 650. Light path 644corresponds to light path 610 positioned in the constricted portion ofthe tapered-to-constricted portion of the rotating growth vial 600, andlight path 650 corresponds to light path 608 in the tapered portion ofthe tapered-to-constricted portion of the rotating growth via 6001.Light paths 610 and 608 are not shown in FIG. 6C but may be seen in FIG.6A. In addition to light paths 644 and 640, there is an emission board648 to illuminate the light path(s), and detector board 646 to detectthe light after the light travels through the cell culture liquid in therotating growth vial 600.

The motor 638 engages with drive mechanism 612 and is used to rotate therotating growth vial 600. In some embodiments, motor 638 is a brushlessDC type drive motor with built-in drive controls that can be set to holda constant revolution per minute (RPM) between 0 and about 3000 RPM.Alternatively, other motor types such as a stepper, servo, brushed DC,and the like can be used. Optionally, the motor 638 may also havedirection control to allow reversing of the rotational direction, and atachometer to sense and report actual RPM. The motor is controlled by aprocessor (not shown) according to, e.g., standard protocols programmedinto the processor and/or user input, and the motor may be configured tovary RPM to cause axial precession of the cell culture thereby enhancingmixing, e.g., to prevent cell aggregation, increase aeration, andoptimize cellular respiration.

Main housing 636, end housings 652 and lower housing 632 of the cellgrowth device 630 may be fabricated from any suitable, robust materialincluding aluminum, stainless steel, and other thermally conductivematerials, including plastics. These structures or portions thereof canbe created through various techniques, e.g., metal fabrication,injection molding, creation of structural layers that are fused, etc.Whereas the rotating growth vial 600 is envisioned in some embodimentsto be reusable, but preferably is consumable, the other components ofthe cell growth device 630 are preferably reusable and function as astand-alone benchtop device or as a module in a multi-module cellprocessing system.

The processor (not shown) of the cell growth device 630 may beprogrammed with information to be used as a “blank” or control for thegrowing cell culture. A “blank” or control is a vessel containing cellgrowth medium only, which yields 100% transmittance and 0 OD, while thecell sample will deflect light rays and will have a lower percenttransmittance and higher OD. As the cells grow in the media and becomedenser, transmittance will decrease and OD will increase. The processor(not shown) of the cell growth device 630—may be programmed to usewavelength values for blanks commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells, etc.). Alternatively, asecond spectrophotometer and vessel may be included in the cell growthdevice 630, where the second spectrophotometer is used to read a blankat designated intervals.

FIG. 6D illustrates a cell growth device 630 as part of an assemblycomprising the cell growth device 630 of FIG. 6B coupled to light source690, detector 692, and thermal components 694. The rotating growth vial600 is inserted into the cell growth device. Components of the lightsource 690 and detector 692 (e.g., such as a photodiode with gaincontrol to cover 5-log) are coupled to the main housing of the cellgrowth device. The lower housing 632 that houses the motor that rotatesthe rotating growth vial 600 is illustrated, as is one of the flanges634 that secures the cell growth device 630 to the assembly. Also, thethermal components 694 illustrated are a Peltier device orthermoelectric cooler. In this embodiment, thermal control isaccomplished by attachment and electrical integration of the cell growthdevice 630 to the thermal components 694 via the flange 634 on the baseof the lower housing 632. Thermoelectric coolers are capable of“pumping” heat to either side of a junction, either cooling a surface orheating a surface depending on the direction of current flow. In oneembodiment, a thermistor is used to measure the temperature of the mainhousing and then, through a standard electronicproportional-integral-derivative (PID) controller loop, the rotatinggrowth vial 600 is controlled to approximately +/−0.5° C.

In use, cells are inoculated (cells can be pipetted, e.g., from anautomated liquid handling system or by a user) into pre-filled growthmedia of a rotating growth vial 600 by piercing though the foil seal orfilm. The programmed software of the cell growth device 630 sets thecontrol temperature for growth, typically 30° C., then slowly starts therotation of the rotating growth vial 600. The cell/growth media mixtureslowly moves vertically up the wall due to centrifugal force allowingthe rotating growth vial 600 to expose a large surface area of themixture to a normal oxygen environment. The growth monitoring systemtakes either continuous readings of the OD or OD measurements at pre-setor pre-programmed time intervals. These measurements are stored ininternal memory and if requested the software plots the measurementsversus time to display a growth curve. If enhanced mixing is required,e.g., to optimize growth conditions, the speed of the vial rotation canbe varied to cause an axial precession of the liquid, and/or a completedirectional change can be performed at programmed intervals. The growthmonitoring can be programmed to automatically terminate the growth stageat a pre-determined OD, and then quickly cool the mixture to a lowertemperature to inhibit further growth.

One application for the cell growth device 630 is to constantly measurethe optical density of a growing cell culture. One advantage of thedescribed cell growth device is that optical density can be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 minutes. While the cell growth device 630 has been describedin the context of measuring the optical density (OD) of a growing cellculture, it should, however, be understood by a skilled artisan giventhe teachings of the present specification that other cell growthparameters can be measured in addition to or instead of cell culture OD.As with optional measure of cell growth in relation to the solid walldevice or module described supra, spectroscopy using visible, UV, ornear infrared (NIR) light allows monitoring the concentration ofnutrients and/or wastes in the cell culture and other spectroscopicmeasurements may be made; that is, other spectral properties can bemeasured via, e.g., dielectric impedance spectroscopy, visiblefluorescence, fluorescence polarization, or luminescence. Additionally,the cell growth device 630 may include additional sensors for measuring,e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like.

Cell Concentration Module

Included in the automated multi-module cell processing instrumentdepicted in FIGS. 5A-5D, is a cell concentration module 522. FIGS. 7A-7Hdepict variations of one embodiment of a cell concentration/bufferexchange module that utilizes tangential flow filtration. The cellconcentration module described herein operates using tangential flowfiltration (TFF), also known as cross-flow filtration, in which themajority of the feed flows tangentially over the surface of the filterthereby reducing cake (retentate) formation as compared to dead-endfiltration, in which the feed flows into the filter. Secondary flowsrelative to the main feed are also exploited to generate shear forcesthat prevent filter cake formation and membrane fouling thus maximizingparticle recovery, as described below.

The TFF device described herein was designed to take into account twoprimary design considerations. First, the geometry of the TFF deviceleads to filtering the cell culture over a large surface area so as tominimize processing time. Second, the design of the TFF device isconfigured to minimize filter fouling. FIG. 7A is a general model 750 oftangential flow filtration. The TFF device operates using tangentialflow filtration, also known as cross-flow filtration. FIG. 7A showscells flowing over a membrane 754, where the feed flow of the cells 752in medium or buffer is parallel to the membrane 754. TFF is differentfrom dead-end filtration where both the feed flow and the pressure dropare perpendicular to a membrane or filter.

FIG. 7B depicts a top view of a lower member 720 of one embodiment of anexemplary TFF device/module providing tangential flow filtration. As canbe seen in FIG. 7B, the lower member 720 of the TFF device modulecomprises a channel structure 716 comprising a flow channel throughwhich a cell culture is flowed. The channel structure 716 comprises asingle flow channel 702 b. (Note, that the flow channel generally isdesignated 702, the portion of the flow channel in the upper member 722of the TFF device is designated 702 a, and the portion of the flowchannel in the lower member 720 of the TFF device is designated 702 b.)This particular embodiment comprises a channel configuration 714, e.g.,an undulating serpentine geometry (i.e., the small “wiggles” in the flowchannel 702) and a serpentine “zig-zag” pattern where the flow channel702 b crisscrosses the lower member 720 of the TFF device from one endat the left of the device to the other end at the right of the device.The serpentine pattern allows for filtration over a high surface arearelative to the device size and total channel volume, while theundulating contribution creates a secondary inertial flow to enableeffective membrane regeneration preventing membrane fouling. Although anundulating geometry and serpentine pattern are exemplified here, otherchannel configurations 714 may be used as long as the flow channel 702can be bifurcated by a membrane as discussed below, and as long as thechannel configuration 714 provides for cell flow through the TFF modulein alternating directions. Portals 704 and 706 are part of channelstructure 716 by operation of the cells passing through flow channel702. Generally, portals 704 collect cells passing through the flowchannel 702 on one side of a membrane (not shown) (the “retentate”), andportals 706 collect the medium (“filtrate” or “permeate”) passingthrough the flow channel 702 on the opposite side of the membrane (notshown). In this embodiment, recesses 708 accommodate screws or otherfasteners (not shown) that allow the components of the TFF device to besecured to one another.

The length 710 and width 712 of the channel structure 716 may varydepending on the volume of the cell culture to be grown and the opticaldensity of the cell culture to be concentrated. The length 710 of thechannel structure 716 typically is from 1 mm to 300 mm, or from 50 mm to250 mm, or from 60 mm to 200 mm, or from 70 mm to 150 mm, or from 80 mmto 100 mm. The width 712 of the channel structure 716 typically is from1 mm to 120 mm, or from 20 mm to 100 mm, or from 30 mm to 80 mm, or from40 mm to 70 mm, or from 50 mm to 60 mm. The cross-section configurationof the flow channel 702 may be round, elliptical, oval, square,rectangular, trapezoidal, or irregular. If square, rectangular, oranother shape with generally straight sides, the cross section may befrom about 10 μm to 1000 μm wide, or from 200 μm to 800 μm wide, or from300 μm to 700 μm wide, or from 400 μm to 600 μm wide; and from about 10μm to 1000 μm high, or from 200 μm to 800 μm high, or from 300 μm to 700μm high, or from 400 μm to 600 μm high. If the cross section of the flowchannel 702 is generally round, oval or elliptical, the radius of thechannel may be from about 50 μm to 1000 μm in hydraulic radius, or from5 μm to 800 μm in hydraulic radius, or from 200 μm to 700 μm inhydraulic radius, or from 300 μm to 600 μm wide in hydraulic radius, orfrom about 200 to 500 μm in hydraulic radius.

When looking at the top view of lower member 720 of the TFFdevice/module of FIG. 7B, note that there are two retentate portals 704and two filtrate portals 706, where there is one of each type of portalat both ends (e.g., the narrow edge) of the TFF device/module 700. Inother embodiments, retentate and filtrate portals can be on the samesurface of the same member (e.g., upper member 722 or lower member 720),or they can be arranged on the side surfaces of the assembly. Unlikeother tangential flow filtration devices that operate continuously, theTFF device/module described herein uses an alternating flow method forconcentrating cells. FIG. 7C depicts a top view of upper (722) and lower(720) members of an exemplary TFF module 700. The lower portion of theflow channel 702 b is seen on the top surface of lower member 720.Again, portals 704 and 706 are seen. As noted above, recesses—such asthe recesses 708 seen in FIG. 7B—provide a means to secure thecomponents (upper member 722, lower member 720, and membrane 724) of theTFF device/membrane 700 to one another during operation via, e.g.,screws or other like fasteners. However, in alternative embodiments, anadhesive—such as a pressure sensitive adhesive—or ultrasonic welding, orsolvent bonding, may be used to couple the upper member 722, lowermember 720, and membrane 724 together. Indeed, one of ordinary skill inthe art given the guidance of the present disclosure can find yet otherconfigurations for coupling the components of the TFF device 700, suchas e.g., clamps; mated fittings disposed on the upper (722) and lower(720) members; combination of adhesives, welding, solvent bonding, andmated fittings; and other such fasteners and couplings.

Note that in FIG. 7C there is one retentate portal 704 and one filtrateportal 706 on each “end” (e.g., the narrow edges) of the TFFdevice/module 700. The retentate 704 and filtrate 706 portals on theleft side of the TFF device/module 700 will collect cells (flow path at760 from the upper member 722) and medium (flow path at 770 from thelower member 720), respectively, when the cells and medium flow fromright to left in the TFF module 700. Likewise, the retentate 704 andfiltrate 706 portals on the right side of the TFF device/module 700 willcollect cells (flow path at 760) and medium (flow path at 770),respectively, when the cells and medium flow from left to right in theTFF device. In this embodiment, the retentate is collected from portals704 on the top surface of the TFF device, and filtrate is collected fromportals 706 on the bottom surface of the device. The cells aremaintained in the TFF flow channel 702 a (shown in FIG. 7D) above themembrane 724, while the filtrate (medium) flows through membrane 724 andthen through filtrate portals 706; thus, the top/retentate portals 704and bottom/filtrate portals 706 configuration is practical. It should berecognized, however, that other configurations of retentate 704 andfiltrate 706 portals may be implemented such as positioning both theretentate 704 and filtrate 706 portals on the side surface (as opposedto the top and bottom surfaces) of the TFF device 700. In FIG. 7C, theflow channel 702 b can be seen on the lower member 720 of the TFF device700. However, in other embodiments, retentate 704 and filtrate 706portals can reside on the same surface of the TFF device.

The overall work flow for cell concentration using the TFF device/module700 involves flowing a cell culture or cell sample tangentially throughthe channel structure 716. The membrane 724 bifurcating the flowchannels 702 retains the cells on one side of the membrane and allowsunwanted medium or buffer to flow across the membrane into the filtrateside (e.g, lower member 720) of the TFF device 700. In this process, afixed volume of cells in medium or buffer is driven through the deviceuntil the cell sample is collected into one of the retentate portals704, and the medium/buffer that has passed through the membrane iscollected through one or both of the filtrate portals 706. All types ofprokaryotic and eukaryotic cells—both adherent and non-adherentcells—can be concentrated in the TFF device. Adherent cells may be grownon beads or other cell scaffolds suspended in medium in the rotatinggrowth vial, then passed through the TFF device.

In the cell concentration process, passing the cell sample through theTFF device and collecting the cells in one of the retentate portals 404while collecting the medium in one of the filtrate portals 706 isconsidered “one pass” of the cell sample. The transfer between retentatereservoirs “flips” the culture. The retentate and filtrate portalscollecting the cells and medium, respectively, for a given pass resideon the same end of TFF device/module 700 with fluidic connectionsarranged so that there are two distinct flow layers (not shown) for theretentate and filtrate sides, but if the retentate portal 404 resides onthe upper member 722 of the TFF device/module 700 (that is, the cellsare driven through the flow channel 702 a (not shown) above the membraneand the filtrate (medium) passes to the portion of the flow channel 702b below the membrane), the filtrate portal 706 will reside on the lowermember of TFF device/module 700 and vice versa (that is, if the cellsample is driven through the flow channel 702 b below the membrane, thefiltrate (medium) passes to the portion of the flow channel 702 a abovethe membrane). This configuration can be seen more clearly in FIGS.7C-7D, where the retentate flow path 760 from the retentate portals 704and the filtrate flow path 770 from the filtrate portals 706.

At the conclusion of a “pass”, the cell sample is collected by passingthrough the retentate portal 704 and into the retentate reservoir (notshown). To initiate another “pass”, the cell sample is passed againthrough the TFF device 700, this time in a flow direction that isreversed from the first pass. The cell sample is collected by passingthrough the retentate portal 704 and into retentate reservoir (notshown) on the opposite end of the TFF device/module 700 from theretentate portal 704 that was used to collect cells during the firstpass. Likewise, the medium/buffer that passes through the membrane onthe second pass is collected through the filtrate portal 706 on theopposite end of the TFF device/module 700 from the filtrate portal 706that was used to collect the filtrate during the first pass, or throughboth portals. This alternating process of passing the retentate (theconcentrated cell sample) through the TFF device/module 700 is repeateduntil the cells have been concentrated to a desired volume, and bothfiltrate portals 706 can be open during the passes to reduce operatingtime. In addition, buffer exchange may be effected by adding a desiredbuffer (or fresh medium) to the cell sample in the retentate reservoir,before initiating another “pass”, and repeating this process until theold medium or buffer is diluted and filtered out and the cells reside infresh medium or buffer. Note that buffer exchange and cell concentrationmay (and typically do) take place simultaneously.

Returning to FIG. 7C, also seen is membrane or filter 724. Filters ormembranes appropriate for use in the TFF device/module 700 are thosethat are solvent resistant, are contamination free during filtration,and are able to retain the types and sizes of cells of interest. Forexample, in order to retain small cell types such as bacterial cells,pore sizes can be as low as 0.2 μm, however for other cell types, thepore sizes can be as high as 5 μm. Indeed, the pore sizes useful in theTFF device/module 700 include filters 724 with sizes from 0.20 μm, 0.21μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm, 0.37μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, 0.45μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. The filters724 may be fabricated from any suitable non-reactive material includingcellulose mixed ester (cellulose nitrate and acetate) (CME),polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone(PES), polytetrafluoroethylene (PTFE), nylon, glass fiber, or metalsubstrates as in the case of laser or electrochemical etching. The TFFdevice 700 shown in FIGS. 7C and 7D does not show a seat in the upper722 and lower 720 members where the filter 724 can be seated or secured(for example, a seat half the thickness of the filter 724 in each ofupper 722 and lower 720 members); however, such a seat is contemplatedin some embodiments.

FIG. 7D depicts a bottom-up view of upper member 722 and lower member720 of the exemplary TFF module shown in FIG. 7C. Again portals 704 and706 are seen. Note again that there is one retentate portal 704 and onefiltrate portal 706 on each end of the upper member 722 and lower member720 of the TFF device/module 700 (not all portals are seen in thisview). On the left side of the TFF device 700, the retentate portals 704will collect cells (flow path at 760) and the filtrate portals 706 willcollect medium (flow path at 770), respectively, for the same pass.Likewise, on the right side of the TFF device 700, the retentate portals704 will collect cells (flow path at 760) and the filtrate portals 706will collect medium (flow path at 770), respectively, for the same pass.In FIG. 7D, the upper portion of flow channel 702 a can be seen on thelower surface of upper member 722 of the TFF device 700. Thus, lookingat FIGS. 7C and 7D, note that there is a flow channel 702 in both theupper member 722 (flow channel 702 a in FIG. 7D) and lower member 722(flow channel 702 b in FIG. 7C) with a membrane 724 between the upper722 and lower 720 members. Again, the flow channels 702 a and 702 b ofthe upper 722 and lower 720 members mate to create the flow channel 702with the membrane 724 positioned horizontally between the upper andlower members of the TFF device/module thereby bifurcating the flowchannel 702 (not shown).

Buffer exchange during cell concentration and/or rendering the cellscompetent is performed on the TFF device/module 700 by adding a desiredbuffer to the cells concentrated to a desired volume; for example, afterthe cells have been concentrated at least 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold,200-fold or more. A desired exchange medium or exchange buffer is addedto the cells by addition to a retentate reservoir (not shown) and theprocess of passing the cells through the TFF device 700 is repeateduntil the cells have been concentrated to a desired volume in theexchange medium or buffer. This process can be repeated any number ofdesired times so as to achieve a desired level of exchange of the bufferand a desired volume of cells. The exchange buffer may comprise, e.g.,glycerol or sorbitol thereby rendering the cells competent fortransformation in addition to decreasing the overall volume of the cellsample.

The TFF device 700 may be fabricated from any robust material in whichflow channels may be milled including stainless steel, silicon, glass,aluminum, or plastics including cyclic-olefin copolymer (COC),cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride, polyamide,polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA),polysulfone, and polyurethane, and co-polymers of these and otherpolymers. If the TFF device/module 700 is disposable, preferably it ismade of plastic. In some embodiments, the material used to fabricate theTFF device/module 700 is thermally-conductive so that the cell culturemay be heated or cooled to a desired temperature. In certainembodiments, the TFF device 700 is formed by precision mechanicalmachining, laser machining, electro discharge machining (for metaldevices); wet or dry etching (for silicon devices); dry or wet etching,powder or sandblasting, photostructuring (for glass devices); orthermoforming, injection molding, hot embossing, or laser machining (forplastic devices) using the materials mentioned above that are amenableto these mass production techniques.

FIG. 7E depicts an exploded perspective view of one exemplary embodimentof a TFF module having fluidically coupled reservoirs for retentate,filtrate, and exchange buffer. In this configuration, 790 is the top orcover of the TFF device, having three ports 766, where there is apipette tip 768 disposed in the left-most port 766. The top 790 of theTFF device is, in operation, coupled with a combined reservoir and uppermember structure 764. Combined reservoir and upper member structure 764comprises a top surface that, in operation, is adjacent the top or cover790 of the TFF device, a bottom surface which comprises the upper member722 of the TFF device, where the upper member 722 of the TFF devicedefines the upper portion of the tangential flow channel (not shown).Combined reservoir and upper member structure 764 comprises tworetentate reservoirs 772 and buffer or medium reservoir 774. Theretentate reservoirs 772 are fluidically coupled to the upper portion ofthe flow channel, and the buffer or medium reservoir 774 is fluidicallycoupled to the retentate reservoirs 772. Also seen in this exploded viewof the TFF device is lower member 720 which, as described previously,comprises on its top surface the lower portion of the tangential flowchannel 702 b (seen on the top surface of lower member 720), where theupper and lower portions of the flow channel 702 of the upper member 722and lower member 720, respectively, when coupled mate to form a singleflow channel 702 (the membrane that is interposed between the uppermember 722 and lower member 720 in operation is not shown).

Beneath lower member 720 is gasket 792, which in operation is interposedbetween lower member 720 and a filtrate (or permeate) reservoir 762. Inoperation, top 790, combined reservoir and upper member structure 764,membrane (not shown), lower member 720, gasket 792, and filtratereservoir 762 are coupled and secured together to be fluid- andair-tight. In FIG. 7E, fasteners are shown that can be used to couplethe various structures (top 790, combined reservoir and upper memberstructure 764, membrane (not shown), lower member 720, gasket 792, andfiltrate reservoir 762) together. However, as an alternative to screwsor other like fasteners, the various structures of the TFF device can becoupled using an adhesive, such as a pressure sensitive adhesive;ultrasonic welding; or solvent bonding. Further, a combination offasteners, adhesives, and/or welding types may be employed to couple thevarious structures of the TFF device. One of ordinary skill in the artgiven the guidance of the present disclosure could find yet otherconfigurations for coupling the components of the TFF device, such as,e.g., clamps, mated fittings, and other such fasteners.

FIG. 7F depicts combined reservoir and upper member structure 764,comprising two retentate reservoirs 772 and buffer or medium reservoir774, as well as upper member 722, which is disposed on the bottom ofcombined reservoir and upper member structure 764. Upper member 722 ofthe TFF device defines the upper portion of the tangential flow channel(not shown) disposed on the bottom surface of the combined reservoir andupper member structure 764. FIG. 7G is a top-down view of the uppersurface 778 of combined reservoir and upper member structure 764,depicting the top 780 of retentate reservoirs 772 and the top 782 ofbuffer or medium reservoir 774. The retentate reservoirs 772 arefluidically coupled to the upper portion of the flow channel (notshown), and the buffer or medium reservoir 774 is fluidically coupled tothe retentate reservoirs 772. FIG. 7H is a bottom-up view of the lowersurface of combined reservoir and upper member structure 764, showingthe upper member 722 with the upper portion of the tangential flowchannel 702 a disposed on the bottom surface of upper member 722. Theflow channel 702 a disposed on the bottom surface of upper member 722 inoperation is mated to the bottom portion of the tangential flow channel702 b disposed on the top surface of the lower member 720 (not shown inthis view, but see FIG. 7E), where the upper and lower portions of theflow channels 702 a and 702 b, respectively, mate to form a single flowchannel 702 with a membrane or filter (not shown) interposed between theupper 702 a and lower 702 b portions of the flow channel.

Nucleic Acid Assembly Module

Certain embodiments of the automated multi-module cell editinginstruments of the present disclosure optionally include a nucleic acidassembly module. The nucleic acid assembly module is configured toaccept and assemble the nucleic acids necessary to facilitate thedesired genome editing events. In general, the term “vector” refers to anucleic acid molecule capable of transporting a desired nucleic acid towhich it has been linked into a cell. Vectors include, but are notlimited to, nucleic acid molecules that are single-stranded,double-stranded, or partially double-stranded; nucleic acid moleculesthat include one or more free ends, no free ends (e.g., circular);nucleic acid molecules that include DNA, RNA, or both; and othervarieties of polynucleotides known in the art. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,where virally-derived DNA or RNA sequences are present in the vector forpackaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g. bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors” or “editingvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. Additional vectors includefosmids, phagemids, and synthetic chromosomes.

Recombinant expression vectors can include a nucleic acid in a formsuitable for transcription, and for some nucleic acid sequences,translation and expression of the nucleic acid in a host cell, whichmeans that the recombinant expression vectors include one or moreregulatory elements—which may be selected on the basis of the host cellsto be used for expression—that are operatively-linked to the nucleicacid sequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory element(s) in a manner that allowsfor transcription, and for some nucleic acid sequences, translation andexpression of the nucleotide sequence (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). Appropriate recombination and cloningmethods are disclosed in US Pub. No. 2004/0171156, the contents of whichare herein incorporated by reference in their entirety for all purposes.

In some embodiments, a regulatory element is operably linked to one ormore elements of a targetable nuclease system so as to drivetranscription, and for some nucleic acid sequences, translation andexpression of the one or more components of the targetable nucleasesystem.

In addition, the polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcells, such as prokaryotic or eukaryotic cells. Eukaryotic cells can beyeast, fungi, algae, plant, animal, or human cells. Eukaryotic cells maybe those of or derived from a particular organism, such as a mammal,including but not limited to human, mouse, rat, rabbit, dog, ornon-human mammal including non-human primate. In addition oralternatively, a vector may include a regulatory element operably likedto a polynucleotide sequence, which, when transcribed, forms a guideRNA.

The nucleic acid assembly module can be configured to perform a widevariety of different nucleic acid assembly techniques in an automatedfashion. Nucleic acid assembly techniques that can be performed in thenucleic acid assembly module of the disclosed automated multi-modulecell editing instruments include, but are not limited to, those assemblymethods that use restriction endonucleases, including PCR, BioBrickassembly (U.S. Pat. No. 9,361,427), Type IIS cloning (e.g., GoldenGateassembly, European Patent Application Publication EP 2 395 087 A1), andLigase Cycling Reaction (de Kok, ACS Synth Biol., 3(2):97-106 (2014);Engler, et al., PLoS One, 3(11):e3647 (2008); and U.S. Pat. No.6,143,527). In other embodiments, the nucleic acid assembly techniquesperformed by the disclosed automated multi-module cell editinginstruments are based on overlaps between adjacent parts of the nucleicacids, such as Gibson Assembly®, CPEC, SLIC, Ligase Cycling etc.Additional assembly methods include gap repair in yeast (Bessa, Yeast,29(10):419-23 (2012)), gateway cloning (Ohtsuka, Curr Pharm Biotechnol,10(2):244-51 (2009)); U.S. Pat. Nos. 5,888,732; and 6,277,608), andtopoisomerase-mediated cloning (Udo, PLoS One, 10(9):e0139349 (2015);and U.S. Pat. No. 6,916,632). These and other nucleic acid assemblytechniques are described, e.g., in Sands and Brent, Curr Protoc MolBiol., 113:3.26.1-3.26.20 (2016).

The nucleic acid assembly module is temperature controlled dependingupon the type of nucleic acid assembly used in the automatedmulti-module cell editing instrument. For example, when PCR is utilizedin the nucleic acid assembly module, the module includes a thermocyclingcapability allowing the temperatures to cycle between denaturation,annealing and extension steps. When single temperature assembly methods(e.g., isothermal assembly methods) are utilized in the nucleic acidassembly module, the module provides the ability to reach and hold atthe temperature that optimizes the specific assembly process beingperformed. These temperatures and the duration for maintaining thesetemperatures can be determined by a preprogrammed set of parametersexecuted by a script, or manually controlled by the user using theprocessing system of the automated multi-module cell editing instrument.

In one embodiment, the nucleic acid assembly module is a module toperform assembly using a single, isothermal reaction. Certain isothermalassembly methods can combine simultaneously up to 15 nucleic acidfragments based on sequence identity. The assembly method provides, insome embodiments, nucleic acids to be assembled which include anapproximate 20-40 base overlap with adjacent nucleic acid fragments. Thefragments are mixed with a cocktail of three enzymes—an exonuclease, apolymerase, and a ligase—along with buffer components. Because theprocess is isothermal and can be performed in a 1-step or 2-step methodusing a single reaction vessel, isothermal assembly reactions are idealfor use in an automated multi-module cell editing instrument. The 1-stepmethod allows for the assembly of up to five different fragments using asingle step isothermal process. The fragments and the master mix ofenzymes are combined and incubated at 50° C. for up to one hour. For thecreation of more complex constructs with up to fifteen fragments or forincorporating fragments from 100 bp up to 10 kb, typically the 2-step isused, where the 2-step reaction requires two separate additions ofmaster mix; one for the exonuclease and annealing step and a second forthe polymerase and ligation steps.

Transformation Module

FIGS. 8A-8E depict variations on one embodiment of a cell transformationmodule (in this case, a flow-through electroporation device) that may beincluded in a cell growth/concentration/transformation instrument. FIGS.8A and 8B are top perspective and bottom perspective views,respectively, of six co-joined flow-through electroporation devices 850.FIG. 8A depicts six flow-through electroporation units 850 arranged on asingle substrate 856. Each of the six flow-through electroporation units850 have inlet wells 852 that define cell sample inlets and outlet wells854 that define cell sample outlets. FIG. 8B is a bottom perspectiveview of the six co-joined flow-through electroporation devices of FIG.8A also depicting six flow-through electroporation units 850 arranged ona single substrate 856. Six inlet wells 852 can be seen, one for eachflow-through electroporation unit 850, and one outlet well 854 can beseen (the outlet well of the left-most flow-through electroporation unit850). Additionally seen in FIG. 8B are an inlet 802, outlet 804, flowchannel 806 and two electrodes 808 on either side of a constriction inflow channel 806 in each flow-through electroporation unit 850. Once thesix flow-through electroporation units 850 are fabricated, they can beseparated from one another (e.g., “snapped apart”) and used one at atime, or alternatively in embodiments where two or more flow-throughelectroporation units 850 can be used in parallel without separation.

The flow-through electroporation devices 850 achieve high efficiencycell electroporation with low toxicity. The flow-through electroporationdevices 850 of the disclosure allow for particularly easy integrationwith robotic liquid handling instrumentation that is typically used inautomated systems such as air displacement pipettors. Such automatedinstrumentation includes, but is not limited to, off-the-shelf automatedliquid handling systems from Tecan (Mannedorf, Switzerland), Hamilton(Reno, Nev.), Beckman Coulter (Fort Collins, Colo.), etc.

Generally speaking, microfluidic electroporation—using cell suspensionvolumes of less than approximately 10 mL and as low as 1 μl—allows moreprecise control over a transfection or transformation process andpermits flexible integration with other cell processing tools comparedto bench-scale electroporation devices. Microfluidic electroporationthus provides unique advantages for, e.g., single cell transformation,processing and analysis; multi-unit electroporation deviceconfigurations; and integrated, automatic, multi-module cell processingand analysis.

In specific embodiments of the flow-through electroporation devices 850of the disclosure, the toxicity level of the transformation results ingreater than 10% viable cells after electroporation, preferably greaterthan 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%,85%, 90%, or even 95% viable cells following transformation, dependingon the cell type and the nucleic acids being introduced into the cells.

The flow-through electroporation device 850 described in relation toFIGS. 8A-8E comprises a housing with an electroporation chamber, a firstelectrode and a second electrode configured to engage with an electricpulse generator, by which electrical contacts engage with the electrodesof the electroporation device 850. In certain embodiments, theelectroporation devices are autoclavable and/or disposable, and may bepackaged with reagents in a reagent cartridge. The electroporationdevice 850 may be configured to electroporate cell sample volumesbetween 1 μl to 2 mL, 10 μl to 1 mL, 25 μl to 750 μl, or 50 μl to 500μl. The cells that may be electroporated with the disclosedelectroporation devices 850 include mammalian cells (including humancells), plant cells, yeasts, other eukaryotic cells, bacteria, archaea,and other cell types.

In one exemplary embodiment, FIG. 8C depicts a top view of aflow-through electroporation device 850 having an inlet 802 forintroduction of cells and an exogenous reagent to be electroporated intothe cells (“cell sample”) and an outlet 804 for the cell samplefollowing electroporation. Electrodes 808 are introduced throughelectrode channels (not shown in this FIG. 8C) in the device. FIG. 8Dshows a cutaway view from the top of flow-through electroporation device850, with the inlet 802, outlet 804, and electrodes 808 positioned withrespect to a constriction in flow channel 806. A side cutaway view of alower portion of flow-through electroporation device 850 in FIG. 8Eillustrates that electrodes 808 in this embodiment are positioned inelectrode channels 810 and perpendicular to flow channel 806 such thatthe cell sample flows from the inlet channel 812 through the flowchannel 806 to the outlet channel 814, and in the process the cellsample flows into the electrode channels 810 to be in contact withelectrodes 808. In this aspect, the inlet channel 812, outlet channel814 and electrode channels 810 all originate from the top planar side ofthe device; however, the flow-through electroporation architecturedepicted in FIGS. 8C-8E is but one architecture useful with the reagentcartridges described herein. Additional electrode architectures aredescribed, e.g., in U.S. Ser. No. 16/147,120, filed 24 Sep. 2018; Ser.No. 16/147,865, filed 30 Sep. 2018; and Ser. No. 16/147,871, filed 30Sep. 2018.

Exemplary Workflows

FIG. 9 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising asingulation or substantial singulation/growth/editing and normalizationor cherry-picking module for enrichment or selection of edited cells.The cell processing instrument 900 may include a housing 944, areservoir of cells to be transformed or transfected 902, and a growthmodule (a cell growth device) 904. The cells to be transformed aretransferred from a reservoir to the growth module to be cultured untilthe cells hit a target OD. Once the cells hit the target OD, the growthmodule may cool or freeze the cells for later processing, or the cellsmay be transferred to a cell concentration module 930 where the cellsare rendered electrocompetent and concentrated to a volume optimal forcell transformation. Once concentrated, the cells are then transferredto the electroporation device 908 (e.g., transformation/transfectionmodule, with one exemplary module described above in relation to FIGS.8A-8E). Exemplary electroporation devices of use in the automatedmulti-module cell processing instruments include flow-throughelectroporation devices such as those described in U.S. Ser. No.16/147,120, filed 28 Sep. 2018; Ser. No. 16/147,353, filed 28 Sep. 2018;Ser. No. 16/147,865, filed 30 Sep. 2018; and Ser. No. 16/147,871, filed30 Sep. 2018 all of which are herein incorporated by reference in theirentirety.

In addition to the reservoir for storing the cells 930, the automatedmulti-module cell processing instrument 900 may include a reservoir forstoring editing oligonucleotide cassettes 916 and a reservoir forstoring an expression vector backbone 918. Both the editingoligonucleotide cassettes and the expression vector backbone aretransferred from a reagent cartridge to a nucleic acid assembly module920, where the editing oligonucleotide cassettes are inserted into theexpression vector backbone. The assembled nucleic acids may betransferred into an optional purification module 922 for desaltingand/or other purification and/or concentration procedures needed toprepare the assembled nucleic acids for transformation. Alternatively,pre-assembled nucleic acids, e.g., an editing vector, may be storedwithin reservoir 916 or 918. Once the processes carried out by thepurification module 922 are complete, the assembled nucleic acids aretransferred to, e.g., an electroporation device 908, which alreadycontains the cell culture grown to a target OD and renderedelectrocompetent via cell concentration module 930. In electroporationdevice 908, the assembled nucleic acids are introduced into the cells.Following electroporation, the cells are transferred into a combinedrecovery/selection module 910. For examples of multi-module cell editinginstruments, see U.S. Ser. Nos. 16/024,816 and 16/024,831, filed 30 Jun.2018; Ser. No. 16/147,865, filed 30 Sep. 2018; and Ser. No. 16/147,871,filed 30 Sep. 2018, all of which are herein incorporated by reference intheir entirety.

Following recovery, and, optionally, selection, the cells aretransferred to a singulation or substantial singulation, editing, andgrowth module 940, where the cells are diluted and compartmentalizedsuch that there is an average of one cell per compartment. Oncesubstantially or largely singulated, the cells are allowed to grow for apre-determined number of doublings. Once these initial colonies areestablished, editing is induced and the edited cells are grown toestablish colonies, which are grown to terminal size (e.g., the coloniesare normalized). In some embodiments, editing is induced by one or moreof the editing components being under the control of an induciblepromoter. In some embodiments, the inducible promoter is activated by arise in temperature and “deactivated” by lowering the temperature.Similarly, in embodiments where the solid wall device comprising afilter forming the bottom of the microwell, the solid wall device can betransferred to a plate (e.g., an agar plate or even to liquid medium)comprising a medium with a component that activates or induces editing,then transferred to a medium that deactivates editing. In solid walldevices with solid bottoms, induction of editing can deactivation ofediting can take place by media exchange. Once the colonies are grown toterminal size, the colonies are pooled. Again, singulation orsubstantial singulation overcomes growth bias from unedited cells andgrowth bias resulting from fitness effects of different edits.

The recovery, selection, and singulation/induction/editing andnormalization modules may all be separate, may be arranged and combinedas shown in FIG. 9, or may be arranged or combined in otherconfigurations. In certain embodiments, all of recovery, selection,singulation or substantial singulation, growth, editing, andnormalization are performed in a single module such as on a substratewith nutrient agar, or on the solid wall device described in relation toFIGS. 3A-3E and 4A-4AA. Alternatively, recovery, selection, anddilution, if necessary, are performed in liquid medium in a separatevessel (module), then transferred to thesingulation/growth/induction/editing and normalization module.

Once the normalized cell colonies are pooled, the cells may be stored,e.g., in a storage unit or module 912, where the cells can be kept at,e.g., 4° C. until the cells are retrieved for further study 914.Alternatively, the cells may be used in another round of editing. Themulti-module cell processing instrument 900 is controlled by a processor942 configured to operate the instrument based on user input, asdirected by one or more scripts, or as a combination of user input or ascript. The processor 942 may control the timing, duration, temperature,and operations of the various modules of the instrument 900 and thedispensing of reagents. For example, the processor 942 may cool thecells post-transformation until editing is desired, upon which time thetemperature may be raised to a temperature conducive of genome editingand cell growth. The processor may be programmed with standard protocolparameters from which a user may select, a user may specify one or moreparameters manually or one or more scripts associated with the reagentcartridge may specify one or more operations and/or reaction parameters.In addition, the processor may notify the user (e.g., via an applicationto a smart phone or other device) that the cells have reached the targetOD as well as update the user as to the progress of the cells in thevarious modules in the multi-module system.

The automated multi-module cell processing instrument 900 is anuclease-directed genome editing system and can be used in singleediting systems (e.g., introducing one or more edits to a cellulargenome in a single editing process). The system of FIG. 10, describedbelow, is configured to perform sequential editing, e.g., usingdifferent nuclease-directed systems sequentially to provide two or moregenome edits in a cell; and/or recursive editing, e.g. utilizing asingle nuclease-directed system to introduce sequentially two or moregenome edits in a cell.

FIG. 10 illustrates another embodiment of an automated multi-module cellprocessing instrument configured to perform singulation or substantialsingulation of cells, growth, induction of editing and normalization ofcell colonies. This embodiment depicts an exemplary system that performsrecursive gene editing on a cell population. As with the embodimentshown in FIG. 9, the cell processing instrument 1000 may include ahousing 1044, a reservoir for storing cells to be transformed ortransfected 1002, and a cell growth module (comprising, e.g., a rotatinggrowth vial) 1004. The cells to be transformed are transferred from areservoir to the cell growth module to be cultured until the cells hit atarget OD. Once the cells hit the target OD, the growth module may coolor freeze the cells for later processing or transfer the cells to a cellconcentration module 1060 where the cells are subjected to bufferexchange and rendered electrocompetent, and the volume of the cells maybe reduced substantially. Once the cells have been concentrated to anappropriate volume, the cells are transferred to electroporation device1008. In addition to the reservoir for storing cells, the multi-modulecell processing instrument 1000 includes a reservoir for storing thevector pre-assembled with editing oligonucleotide cassettes 1052. Thepre-assembled nucleic acid vectors are transferred to theelectroporation device 1008, which already contains the cell culturegrown to a target OD. In the electroporation device 1008, the nucleicacids are electroporated into the cells. Following electroporation, thecells are transferred into an optional recovery module 1056, where thecells are allowed to recover briefly post-transformation.

After recovery, the cells may be transferred to a storage unit or module1012, where the cells can be stored at, e.g., 4° C. for laterprocessing, or the cells may be diluted and transferred to a solid wallselection, singulation or substantialsingulation/growth/induction/editing and normalization module 1058. Inthe multi-process module 1058, the cells are arrayed such that there isan average of one cell per microwell. The arrayed cells may be inselection medium to select for cells that have been transformed ortransfected with the editing vector(s). Once substantially or largelysingulated, the cells grow through 2-50 doublings and establishcolonies. Once colonies are established, editing is induced by providingconditions (e.g., temperature, addition of an inducing or repressingchemical) to induce editing. Once editing is initiated and allowed toproceed, the cells are allowed to grow to terminal size (e.g.,normalization of the colonies) in the microwells and then can be flushedout of the microwells and pooled, then transferred to the cell retrievalunit 1014 or can be transferred back to a growth module 1004 for anotherround of editing. In between pooling and transfer to a growth module,there may be one or more additional steps, such as cell recovery, mediumexchange, cells concentration, etc., by, e.g., tangential flowfiltration. Note that the selection/singulation or substantialsingulation/growth/induction/editing and normalization modules may bethe same module, where all processes are performed in the solid walldevice, or selection and/or dilution may take place in a separate vesselbefore the cells are transferred to the solid wall singulation orsubstantial singulation/growth/induction/editing and normalizationmodule (solid wall device). Once the putatively-edited cells are pooled,they may be subjected to another round of editing, beginning withgrowth, cell concentration and treatment to render electrocompetent, andtransformation by yet another donor nucleic acid in another editingcassette via the electroporation module 1008.

In electroporation device 1008, the cells selected from the first roundof editing are transformed by a second set of editing oligos (or othertype of oligos) and the cycle is repeated until the cells have beentransformed and edited by a desired number of, e.g., editing cassettes.The multi-module cell processing instrument 1000 exemplified in FIG. 10is controlled by a processor 1042 configured to operate the instrumentbased on user input or is controlled by one or more scripts including atleast one script associated with the reagent cartridge. The processor1042 may control the timing, duration, and temperature of variousprocesses, the dispensing of reagents, and other operations of thevarious modules of the automated multi-module cell processing instrument1000. For example, a script or the processor may control the dispensingof cells, reagents, vectors, and editing oligonucleotides; which editingoligonucleotides are used for cell editing and in what order; the time,temperature and other conditions used in the recovery and expressionmodule; the wavelength at which OD is read in the cell growth module,the target OD to which the cells are grown, and the target time at whichthe cells will reach the target OD. In addition, the processor may beprogrammed to notify a user (e.g., via an application to a smart phoneor other device) as to the progress of the cells in the automatedmulti-module cell processing instrument.

It should be apparent to one of ordinary skill in the art given thepresent disclosure that the process described may be recursive andmultiplexed; that is, cells may go through the workflow described inrelation to FIG. 10, then the resulting edited culture may go throughanother (or several or many) rounds of additional editing (e.g.,recursive editing) with different editing vectors. For example, thecells from round 1 of editing may be diluted and an aliquot of theedited cells edited by editing vector A may be combined with editingvector B, an aliquot of the edited cells edited by editing vector A maybe combined with editing vector C, an aliquot of the edited cells editedby editing vector A may be combined with editing vector D, and so on fora second round of editing. After round two, an aliquot of each of thedouble-edited cells may be subjected to a third round of editing, where,e.g., aliquots of each of the AB-, AC-, AD-edited cells are combinedwith additional editing vectors, such as editing vectors X, Y, and Z.That is to say that double-edited cells AB may be combined with andedited by vectors X, Y, and Z to produce triple-edited edited cells ABX,ABY, and ABZ; double-edited cells AC may be combined with and edited byvectors X, Y, and Z to produce triple-edited cells ACX, ACY, and ACZ;and double-edited cells AD may be combined with and edited by vectors X,Y, and Z to produce triple-edited cells ADX, ADY, and ADZ, and so on. Inthis process, many permutations and combinations of edits can beexecuted, leading to very diverse cell populations and cell libraries.In any recursive process, it is advantageous to “cure” the previousengine and editing vectors (or single engine+editing vector in a singlevector system). “Curing” is a process in which one or more vectors usedin the prior round of editing is eliminated from the transformed cells.Curing can be accomplished by, e.g., cleaving the vector(s) using acuring plasmid thereby rendering the editing and/or engine vector (orsingle, combined vector) nonfunctional; diluting the vector(s) in thecell population via cell growth (that is, the more growth cycles thecells go through, the fewer daughter cells will retain the editing orengine vector(s)), or by, e.g., utilizing a heat-sensitive origin ofreplication on the editing or engine vector (or combined engine+editingvector). The conditions for curing will depend on the mechanism used forcuring; that is, in this example, how the curing plasmid cleaves theediting and/or engine plasmid.

Production of Cell Libraries Using Automated Editing Methods, Modules,Instruments and Systems

In one aspect, the present disclosure provides automated editingmethods, modules, instruments, and automated multi-module cell editinginstruments for creating a library of cells that vary the expression,levels and/or activity of RNAs and/or proteins of interest in variouscell types using various editing strategies, as described herein in moredetail. Accordingly, the disclosure is intended to cover edited celllibraries created by the automated editing methods, automatedmulti-module cell editing instruments of the disclosure. These celllibraries may have different targeted edits, including but not limitedto gene knockouts, gene knock-ins, insertions, deletions, singlenucleotide edits, short tandem repeat edits, frameshifts, triplet codonexpansion, and the like in cells of various organisms. These edits canbe directed to coding or non-coding regions of the genome and arepreferably rationally designed.

In other aspects, the present disclosure provides automated editingmethods, automated multi-module cell editing instruments for creating alibrary of cells that vary DNA-linked processes. For example, the celllibrary may include individual cells having edits in DNA binding sitesto interfere with DNA binding of regulatory elements that modulateexpression of selected genes. In addition, cell libraries may includeedits in genomic DNA that impact on cellular processes such asheterochromatin formation, switch-class recombination and VDJrecombination.

In specific aspects, the cell libraries are created using multiplexedediting of individual cells within a cell population, with multiplecells within a cell population are edited in a single round of editing,i.e., multiple changes within the cells of the cell library are in asingle automated operation. The libraries that can be created in asingle multiplexed automated operation can comprise as many as 500edited cells, 1000 edited cells, 2000 edited cells, 5000 edited cells,10,000 edited cells, 50,000 edited cells, 100,000 edited cells, 200,000edited cells, 300,000 edited cells, 400,000 edited cells, 500,000 editedcells, 600,000 edited cells, 700,000 edited cells, 800,000 edited cells,900,000 edited cells, 1,000,000 edited cells, 2,000,000 edited cells,3,000,000 edited cells, 4,000,000 edited cells, 5,000,000 edited cells,6,000,000 edited cells, 7,000,000 edited cells, 8,000,000 edited cells,9,000,000 edited cells, 10,000,000 edited cells or more.

In other specific aspects, the cell libraries are created usingrecursive editing of individual cells within a cell population, withedits being added to the individual cells in two or more rounds ofediting. The use of recursive editing results in the amalgamation of twoor more edits targeting two or more sites in the genome in individualcells of the library. The libraries that can be created in an automatedrecursive operation can comprise as many as 500 edited cells, 1000edited cells, 2000 edited cells, 5000 edited cells, 10,000 edited cells,50,000 edited cells, 100,000 edited cells, 200,000 edited cells, 300,000edited cells, 400,000 edited cells, 500,000 edited cells, 600,000 editedcells, 700,000 edited cells, 800,000 edited cells, 900,000 edited cells,1,000,000 edited cells, 2,000,000 edited cells, 3,000,000 edited cells,4,000,000 edited cells, 5,000,000 edited cells, 6,000,000 edited cells,7,000,000 edited cells, 8,000,000 edited cells, 9,000,000 edited cells,10,000,000 edited cells or more.

Examples of non-automated editing strategies that can be modified basedon the present specification to utilize the automated systems can befound, e.g., U.S. Pat. Nos. 8,110,360, 8,332,160, 9,988,624,20170316353, and 20120277120.

In specific aspects, recursive editing can be used to first create acell phenotype, and then later rounds of editing used to reverse thephenotype and/or accelerate other cell properties.

In some aspects, the cell library comprises edits for the creation ofunnatural amino acids in a cell.

In specific aspects, the disclosure provides edited cell librarieshaving edits in one or more regulatory elements created using theautomated editing methods, automated multi-module cell editinginstruments of the disclosure. The term “regulatory element” refers tonucleic acid molecules that can influence the transcription and/ortranslation of an operably linked coding sequence in a particularenvironment and/or context. This term is intended to include allelements that promote or regulate transcription, and RNA stabilityincluding promoters, core elements required for basic interaction of RNApolymerase and transcription factors, upstream elements, enhancers, andresponse elements (see, e.g., Lewin, “Genes V” (Oxford University Press,Oxford) pages 847-873). Exemplary regulatory elements in prokaryotesinclude, but are not limited to, promoters, operator sequences and aribosome binding sites. Regulatory elements that are used in eukaryoticcells may include, but are not limited to, promoters, enhancers,insulators, splicing signals and polyadenylation signals.

Preferably, the edited cell library includes rationally designed editsthat are designed based on predictions of protein structure, expressionand/or activity in a particular cell type. For example, rational designmay be based on a system-wide biophysical model of genome editing with aparticular nuclease and gene regulation to predict how different editingparameters including nuclease expression and/or binding, growthconditions, and other experimental conditions collectively control thedynamics of nuclease editing. See, e.g., Farasat and Salis, PLoS ComputBiol., 29:12(1):e1004724 (2016).

In one aspect, the present disclosure provides the creation of a libraryof edited cells with various rationally designed regulatory sequencescreated using the automated editing instrumentation, systems and methodsof the invention. For example, the edited cell library can includeprokaryotic cell populations created using set of constitutive and/orinducible promoters, enhancer sequences, operator sequences and/orribosome binding sites. In another example, the edited cell library caninclude eukaryotic sequences created using a set of constitutive and/orinducible promoters, enhancer sequences, operator sequences, and/ordifferent Kozak sequences for expression of proteins of interest.

In some aspects, the disclosure provides cell libraries including cellswith rationally designed edits comprising one or more classes of editsin sequences of interest across the genome of an organism. In specificaspects, the disclosure provides cell libraries including cells withrationally designed edits comprising one or more classes of edits insequences of interest across a subset of the genome. For example, thecell library may include cells with rationally designed edits comprisingone or more classes of edits in sequences of interest across the exome,e.g., every or most open reading frames of the genome. For example, thecell library may include cells with rationally designed edits comprisingone or more classes of edits in sequences of interest across the kinome.In yet another example, the cell library may include cells withrationally designed edits comprising one or more classes of edits insequences of interest across the secretome. In yet other aspects, thecell library may include cells with rationally designed edits created toanalyze various isoforms of proteins encoded within the exome, and thecell libraries can be designed to control expression of one or morespecific isoforms, e.g., for transcriptome analysis.

Importantly, in certain aspects the cell libraries may comprise editsusing randomized sequences, e.g., randomized promoter sequences, toreduce similarity between expression of one or more proteins inindividual cells within the library. Additionally, the promoters in thecell library can be constitutive, inducible or both to enable strongand/or titratable expression.

In other aspects, the present disclosure provides automated editingmethods, automated multi-module cell editing instruments for creating alibrary of cells comprising edits to identify optimum expression of aselected gene target. For example, production of biochemicals throughmetabolic engineering often requires the expression of pathway enzymes,and the best production yields are not always achieved by the highestamount of the target pathway enzymes in the cell, but rather byfine-tuning of the expression levels of the individual enzymes andrelated regulatory proteins and/or pathways. Similarly, expressionlevels of heterologous proteins sometimes can be experimentally adjustedfor optimal yields.

The most obvious way that transcription impacts on gene expressionlevels is through the rate of Pol II initiation, which can be modulatedby combinations of promoter or enhancer strength and trans-activatingfactors (Kadonaga, et al., Cell, 116(2):247-57 (2004)). In eukaryotes,elongation rate may also determine gene expression patterns byinfluencing alternative splicing (Cramer et al., PNAS USA,94(21):11456-60 (1997)). Failed termination on a gene can impair theexpression of downstream genes by reducing the accessibility of thepromoter to Pol II (Greger, et al., PNAS USA, 97(15):8415-20 (2000)).This process, known as transcriptional interference, is particularlyrelevant in lower eukaryotes, as they often have closely spaced genes.

In some embodiments the present disclosure provides methods foroptimizing cellular gene transcription. Gene transcription is the resultof several distinct biological phenomena, including transcriptionalinitiation (RNAp recruitment and transcriptional complex formation),elongation (strand synthesis/extension), and transcriptional termination(RNAp detachment and termination).

Site-Directed Mutagenesis

Cell libraries can be created using the automated editing methods,modules, instruments, and systems employing site-directed mutagenesis,i.e., when the amino acid sequence of a protein or other genomic featuremay be altered by deliberately and precisely by mutating the protein orgenomic feature. These cell lines can be useful for various purposes,e.g., for determining protein function within cells, the identificationof enzymatic active sites within cells, and the design of novelproteins. For example, site-directed mutagenesis can be used in amultiplexed fashion to exchange a single amino acid in the sequence of aprotein for another amino acid with different chemical properties. Thisallows one to determine the effect of a rationally designed or randomlygenerated mutation in individual cells within a cell population. See,e.g., Berg, et al. Biochemistry, Sixth Ed. (New York: W.H. Freeman andCompany) (2007).

In another example, edits can be made to individual cells within a celllibrary to substitute amino acids in binding sites, such as substitutionof one or more amino acids in a protein binding site for interactionwithin a protein complex or substitution of one or more amino acids inenzymatic pockets that can accommodate a cofactor or ligand. This classof edits allows the creation of specific manipulations to a protein tomeasure certain properties of one or more proteins, includinginteraction with other cofactors, ligands, etc. within a proteincomplex.

In yet another examples, various edit types can be made to individualcells within a cell library using site specific mutagenesis for studyingexpression quantitative trait loci (eQTLs). An eQTL is a locus thatexplains a fraction of the genetic variance of a gene expressionphenotype. The libraries of the invention would be useful to evaluateand link eQTLs to actual diseased states.

In specific aspects, the edits introduced into the cell libraries of thedisclosure may be created using rational design based on known orpredicted structures of proteins. See, e.g., Chronopoulou and Labrou,Curr Protoc Protein Sci.; Chapter 26:Unit 26.6 (2011). Suchsite-directed mutagenesis can provide individual cells within a librarywith one or more site-directed edits, and preferably two or moresite-directed edits (e.g., combinatorial edits) within a cellpopulation.

In other aspects, cell libraries of the disclosure are created usingsite-directed codon mutation “scanning” of all or substantially all ofthe codons in the coding region of a gene. In this fashion, individualedits of specific codons can be examined for loss-of-function orgain-of-function based on specific polymorphisms in one or more codonsof the gene. These libraries can be a powerful tool for determiningwhich genetic changes are silent or causal of a specific phenotype in acell or cell population. The edits of the codons may be randomlygenerated or may be rationally designed based on known polymorphismsand/or mutations that have been identified in the gene to be analyzed.Moreover, using these techniques on two or more genes in a single in apathway in a cell may determine potential protein:protein interactionsor redundancies in cell functions or pathways.

For example, alanine scanning can be used to determine the contributionof a specific residue to the stability or function of given protein.See, e.g., Lefèvre, et al., Nucleic Acids Research, Volume 25(2):447-448(1997). Alanine is often used in this codon scanning technique becauseof its non-bulky, chemically inert, methyl functional group that canmimic the secondary structure preferences that many of the other aminoacids possess. Codon scanning can also be used to determine whether theside chain of a specific residue plays a significant role in cellfunction and/or activity. Sometimes other amino acids such as valine orleucine can be used in the creation of codon scanning cell libraries ifconservation of the size of mutated residues is needed.

In other specific aspects, cell libraries can be created using theautomated editing methods, automated multi-module cell editinginstruments of the invention to determine the active site of a proteinsuch as an enzyme or hormone, and to elucidate the mechanism of actionof one or more of these proteins in a cell library. Site-directedmutagenesis associated with molecular modeling studies can be used todiscover the active site structure of an enzyme and consequently itsmechanism of action. Analysis of these cell libraries can provide anunderstanding of the role exerted by specific amino acid residues at theactive sites of proteins, in the contacts between subunits of proteincomplexes, on intracellular trafficking and protein stability/half-lifein various genetic backgrounds.

Saturation Mutagenesis

In some aspects, the cell libraries created using the automated editingmethods and automated multi-module cell editing instruments of thedisclosure may be saturation mutagenesis libraries, in which a singlecodon or set of codons is randomized to produce all possible amino acidsat the position of a particular gene or genes of interest. These celllibraries can be particularly useful to generate variants, e.g., fordirected evolution. See, e.g., Chica, et al., Current Opinion inBiotechnology 16 (4): 378-384 (2005); and Shivange, Current Opinion inChemical Biology, 13 (1): 19-25 (2009).

In some aspects, edits comprising different degenerate codons can beused to encode sets of amino acids in the individual cells in thelibraries. Because some amino acids are encoded by more codons thanothers, the exact ratio of amino acids cannot be equal. In certainaspects, more restricted degenerate codons are used. ‘NNK’ and ‘NNS’have the benefit of encoding all 20 amino acids, but still encode a stopcodon 3% of the time. Alternative codons such as ‘NDT’, ‘DBK’ avoid stopcodons entirely, and encode a minimal set of amino acids that stillencompass all the main biophysical types (anionic, cationic, aliphatichydrophobic, aromatic hydrophobic, hydrophilic, small).

In specific aspects, the non-redundant saturation mutagenesis, in whichthe most commonly used codon for a particular organism is used in thesaturation mutagenesis editing process.

Promoter Swaps and Ladders

One mechanism for analyzing and/or optimizing expression of one or moregenes of interest is through the creation of a “promoter swap” celllibrary, in which the cells comprise genetic edits that have specificpromoters linked to one or more genes of interest. Accordingly, the celllibraries created using the methods, automated multi-module cell editinginstruments of the disclosure may be promoter swap cell libraries, whichcan be used, e.g., to increase or decrease expression of a gene ofinterest to optimize a metabolic or genetic pathway. In some aspects,the promoter swap cell library can be used to identify an increase orreduction in the expression of a gene that affects cell vitality orviability, e.g., a gene encoding a protein that impacts on the growthrate or overall health of the cells. In some aspects, the promoter swapcell library can be used to create cells having dependencies and logicbetween the promoters to create synthetic gene networks. In someaspects, the promoter swaps can be used to control cell to cellcommunication between cells of both homogeneous and heterogeneous(complex tissues) populations in nature.

The cell libraries can utilize any given number of promoters that havebeen grouped together based upon exhibition of a range of expressionstrengths and any given number of target genes. The ladder of promotersequences vary expression of at least one locus under at least onecondition. This ladder is then systematically applied to a group ofgenes in the organism using the automated editing methods, automatedmulti-module cell editing instruments of the disclosure.

In specific aspects, the cell library formed using the automated editingprocesses, modules and systems of the disclosure include individualcells that are representative of a given promoter operably linked to oneor more target genes of interest in an otherwise identical geneticbackground. Examples of non-automated editing strategies that can bemodified to utilize the automated systems can be found, e.g., in U.S.Pat. No. 9,988,624.

In specific aspects, the promoter swap cell library is produced byediting a set of target genes to be operably linked to a pre-selectedset of promoters that act as a “promoter ladder” for expression of thegenes of interest. For example, the cells are edited so that one or moreindividual genes of interest are edited to be operably linked with thedifferent promoters in the promoter ladder. When an endogenous promoterdoes not exist, its sequence is unknown, or it has been previouslychanged in some manner, the individual promoters of the promoter laddercan be inserted in front of the genes of interest. These produced celllibraries have individual cells with an individual promoter of theladder operably linked to one or more target genes in an otherwiseidentical genetic context.

The promoters are generally selected to result in variable expressionacross different loci, and may include inducible promoters, constitutivepromoters, or both.

The set of target genes edited using the promoter ladder can include allor most open reading frames (ORFs) in a genome, or a selected subset ofthe genome, e.g., the ORFs of the kinome or a secretome. In someaspects, the target genes can include coding regions for variousisoforms of the genes, and the cell libraries can be designed toexpression of one or more specific isoforms, e.g., for transcriptomeanalysis using various promoters.

The set of target genes can also be genes known or suspected to beinvolved in a particular cellular pathway, e.g. a regulatory pathway orsignaling pathway. The set of target genes can be ORFs related tofunction, by relation to previously demonstrated beneficial edits(previous promoter swaps or previous SNP swaps), by algorithmicselection based on epistatic interactions between previously generatededits, other selection criteria based on hypotheses regarding beneficialORF to target, or through random selection. In specific embodiments, thetarget genes can comprise non-protein coding genes, including non-codingRNAs.

Editing of other functional genetic elements, including insulatorelements and other genomic organization elements, can also be used tosystematically vary the expression level of a set of target genes, andcan be introduced using the methods, automated multi-module cell editinginstruments of the disclosure. In one aspect, a population of cells isedited using a ladder of enhancer sequences, either alone or incombination with selected promoters or a promoter ladder, to create acell library having various edits in these enhancer elements. In anotheraspect, a population of cells is edited using a ladder of ribosomebinding sequences, either alone or in combination with selectedpromoters or a promoter ladder, to create a cell library having variousedits in these ribosome binding sequences.

In another aspect, a population of cells is edited to allow theattachment of various mRNA and/or protein stabilizing or destabilizingsequences to the 5′ or 3′ end, or at any other location, of a transcriptor protein.

In certain aspects, a population of cells of a previously establishedcell line may be edited using the automated editing methods, modules,instruments, and systems of the disclosure to create a cell library toimprove the function, health and/or viability of the cells. For example,many industrial strains currently used for large scale manufacturinghave been developed using random mutagenesis processes iteratively overa period of many years, sometimes decades. Unwanted neutral anddetrimental mutations were introduced into strains along with beneficialchanges, and over time this resulted in strains with deficiencies inoverall robustness and key traits such as growth rates. In anotherexample, mammalian cell lines continue to mutate through the passage ofthe cells over periods of time, and likewise these cell lines can becomeunstable and acquire traits that are undesirable. The automated editingmethods, automated multi-module cell editing instruments of thedisclosure can use editing strategies such as SNP and/or STR swapping,indel creation, or other techniques to remove or change the undesirablegenome sequences and/or introducing new genome sequences to address thedeficiencies while retaining the desirable properties of the cells.

When recursive editing is used, the editing in the individual cells inthe edited cell library can incorporate the inclusion of “landing pads”in an ectopic site in the genome (e.g., a CarT locus) to optimizeexpression, stability and/or control.

In some embodiments, each library produced having individual cellscomprising one or more edits (either introducing or removing) iscultured and analyzed under one or more criteria (e.g., production of achemical or product of interest). The cells possessing the specificcriteria are then associated, or correlated, with one or more particularedits in the cell. In this manner, the effect of a given edit on anynumber of genetic or phenotypic traits of interest can be determined.The identification of multiple edits associated with particular criteriaor enhanced functionality/robustness may lead to cells with highlydesirable characteristics.

Knock-Out or Knock-in Libraries

In certain aspects, the present disclosure provides automated editingmethods, modules, instruments and systems for creating a library ofcells having “knock-out” (KO) or “knock-in” (KI) edits of various genesof interest. Thus, the disclosure is intended to cover edited celllibraries created by the automated editing methods, automatedmulti-module cell editing instruments of the disclosure that have one ormore mutations that remove or reduce the expression of selected genes ofinterest to interrogate the effect of these edits on gene function inindividual cells within the cell library.

The cell libraries can be created using targeted gene KO (e.g., viainsertion/deletion) or KOs (e.g., via homologous directed repair). Forexample, double strand breaks are often repaired via the non-homologousend joining DNA repair pathway. The repair is known to be error prone,and thus insertions and deletions may be introduced that can disruptgene function. Preferably the edits are rationally designed tospecifically affect the genes of interest, and individual cells can becreated having a KI or KI of one or more locus of interest. Cells havinga KO or KI of two or more loci of interest can be created usingautomated recursive editing of the disclosure.

In specific aspects, the KO or KI cell libraries are created usingsimultaneous multiplexed editing of cells within a cell population, andmultiple cells within a cell population are edited in a single round ofediting, i.e., multiple changes within the cells of the cell library arein a single automated operation. In other specific aspects, the celllibraries are created using recursive editing of individual cells withina cell population, and results in the amalgamation of multiple edits oftwo or more sites in the genome into single cells.

SNP or Short Tandem Repeat Swaps

In one aspect, cell libraries are created using the automated editingmethods, automated multi-module cell editing instruments of thedisclosure by systematic introducing or substituting single nucleotidepolymorphisms (“SNPs”) into the genomes of the individual cells tocreate a “SNP swap” cell library. In some embodiments, the SNP swappingmethods of the present disclosure include both the addition ofbeneficial SNPs, and removing detrimental and/or neutral SNPs. The SNPswaps may target coding sequences, non-coding sequences, or both.

In another aspect, a cell library is created using the automated editingmethods, modules, instruments, instruments, and systems of thedisclosure by systematic introducing or substituting short tandemrepeats (“STR”) into the genomes of the individual cells to create an“STR swap” cell library. In some embodiments, the STR swapping methodsof the present disclosure include both the addition of beneficial STRs,and removing detrimental and/or neutral STRs. The STR swaps may targetcoding sequences, non-coding sequences, or both.

In some embodiments, the SNP and/or STR swapping used to create the celllibrary is multiplexed, and multiple cells within a cell population areedited in a single round of editing, i.e., multiple changes within thecells of the cell library are in a single automated operation. In otherembodiments, the SNP and/or STR swapping used to create the cell libraryis recursive, and results in the amalgamation of multiple beneficialsequences and/or the removal of detrimental sequences into single cells.Multiple changes can be either a specific set of defined changes or apartly randomized, combinatorial library of mutations. Removal ofdetrimental mutations and consolidation of beneficial mutations canprovide immediate improvements in various cellular processes. Removal ofgenetic burden or consolidation of beneficial changes into a strain withno genetic burden also provides a new, robust starting point foradditional random mutagenesis that may enable further improvements.

SNP swapping overcomes fundamental limitations of random mutagenesisapproaches as it is not a random approach, but rather the systematicintroduction or removal of individual mutations across cells.

Splice Site Editing

RNA splicing is the process during which introns are excised and exonsare spliced together to create the mRNA that is translated into aprotein. The precise recognition of splicing signals by cellularmachinery is critical to this process. Accordingly, in some aspects, apopulation of cells is edited using a systematic editing to known and/orpredicted splice donor and/or acceptor sites in various loci to create alibrary of splice site variants of various genes. Such editing can helpto elucidate the biological relevance of various isoforms of genes in acellular context. Sequences for rational design of splicing sites ofvarious coding regions, including actual or predicted mutationsassociated with various mammalian disorders, can be predicted usinganalysis techniques such as those found in Nalla and Rogan, Hum Mutat,25:334-342 (2005); Divina, et al., Eur J Hum Genet, 17:759-765 (2009);Desmet, et el., Nucleic Acids Res, 37:e67 (2009); Faber, et al., BMCBioinformatics, 12(suppl 4):S2 (2011).

Start/Stop Codon Exchanges and Incorporation of Nucleic Acid Analogs

In some aspects, the present disclosure provides for the creation ofcell libraries using the automated editing methods, modules, instrumentsand systems of the disclosure, where the libraries are created byswapping start and stop codon variants throughout the genome of anorganism or for a selected subset of coding regions in the genome, e.g.,the kinome or secretome. In the cell library, individual cells will haveone or more start or stop codons replacing the native start or stopcodon for one or more gene of interest.

For example, typical start codons used by eukaryotes are ATG (AUG) andprokaryotes use ATG (AUG) the most, followed by GTG (GUG) and TTG (UUG).The cell library may include individual cells having substitutions forthe native start codons for one or more genes of interest.

In some aspects, the present disclosure provides for automated creationof a cell library by replacing ATG start codons with TTG in front ofselected genes of interest. In other aspects, the present disclosureprovides for automated creation of a cell library by replacing ATG startcodons with GTG. In other aspects, the present disclosure provides forautomated creation of a cell library by replacing GTG start codons withATG. In other aspects, the present disclosure provides for automatedcreation of a cell library by replacing GTG start codons with TTG. Inother aspects, the present disclosure provides for automated creation ofa cell library by replacing TTG start codons with ATG. In other aspects,the present disclosure provides for automated creation of a cell libraryby replacing TTG start codons with GTG.

In other examples, typical stop codons for S. cerevisiae and mammals areTAA (UAA) and TGA (UGA), respectively. The typical stop codon formonocotyledonous plants is TGA (UGA), whereas insects and E. colicommonly use TAA (UAA) as the stop codon (Dalphin. et al., Nucl. AcidsRes., 24: 216-218 (1996)). The cell library may include individual cellshaving substitutions for the native stop codons for one or more genes ofinterest.

In some aspects, the present disclosure provides for automated creationof a cell library by replacing TAA stop codons with TAG. In otheraspects, the present disclosure provides for automated creation of acell library by replacing TAA stop codons with TGA. In other aspects,the present disclosure provides for automated creation of a cell libraryby replacing TGA stop codons with TAA. In other aspects, the presentdisclosure provides for automated creation of a cell library byreplacing TGA stop codons with TAG. In other aspects, the presentdisclosure provides for automated creation of a cell library byreplacing TAG stop codons with TAA. In other aspects, the presentinvention teaches automated creation of a cell library by replacing TAGstop codons with TGA.

Terminator Swaps and Ladders

One mechanism for identifying optimum termination of a pre-spliced mRNAof one or more genes of interest is through the creation of a“terminator swap” cell library, in which the cells comprise geneticedits that have specific terminator sequences linked to one or moregenes of interest. Accordingly, the cell libraries created using themethods, modules, instruments and systems of the disclosure may beterminator swap cell libraries, which can be used, e.g., to affect mRNAstability by releasing transcripts from sites of synthesis. In otherembodiments, the terminator swap cell library can be used to identify anincrease or reduction in the efficiency of transcriptional terminationand thus accumulation of unspliced pre-mRNA (e.g., West and Proudfoot,Mol Cell.; 33(3-9); 354-364 (2009)) and/or 3′ end processing (e.g.,West, et al., Mol Cell. 29(5):600-10 (2008)). In the case where a geneis linked to multiple termination sites, the edits may edit acombination of edits to multiple terminators that are associated with agene. Additional amino acids may also be added to the ends of proteinsto determine the effect on the protein length on terminators.

The cell libraries can utilize any given number of edits of terminatorsthat have been selected for the terminator ladder based upon exhibitionof a range of activity and any given number of target genes. The ladderof terminator sequences vary expression of at least one locus under atleast one condition. This ladder is then systematically applied to agroup of genes in the organism using the automated editing methods,modules, instruments and systems of the disclosure.

In some aspects, the present disclosure provides for the creation ofcell libraries using the automated editing methods, modules, instrumentsand systems of disclosure, where the libraries are created to editterminator signals in one or more regions in the genome in theindividual cells of the library. Transcriptional termination ineukaryotes operates through terminator signals that are recognized byprotein factors associated with the RNA polymerase II. For example, thecell library may contain individual eukaryotic cells with edits in genesencoding polyadenylation specificity factor (CPSF) and cleavagestimulation factor (CstF) and or gene encoding proteins recruited byCPSF and CstF factors to termination sites. In prokaryotes, twoprincipal mechanisms, termed Rho-independent and Rho-dependenttermination, mediate transcriptional termination. For example, the celllibrary may contain individual prokaryotic cells with edits in genesencoding proteins that affect the binding, efficiency and/or activity ofthese termination pathways.

In certain aspects, the present disclosure provides methods of selectingtermination sequences (“terminators”) with optimal properties. Forexample, in some embodiments, the present disclosure teaches providesmethods for introducing and/or editing one or more terminators and/orgenerating variants of one or more terminators within a host cell, whichexhibit a range of activity. A particular combination of terminators canbe grouped together as a terminator ladder, and cell libraries of thedisclosure include individual cells that are representative ofterminators operably linked to one or more target genes of interest inan otherwise identical genetic background. Examples of non-automatedediting strategies that can be modified to utilize the automatedinstruments can be found, e.g., in U.S. Pat. No. 9,988,624 to Serber etal., entitled “Microbial strain improvement by a HTP genomic engineeringplatform.”

In specific aspects, the terminator swap cell library is produced byediting a set of target genes to be operably linked to a pre-selectedset of terminators that act as a “terminator ladder” for expression ofthe genes of interest. For example, the cells are edited so that theendogenous promoter is operably linked to the individual genes ofinterest are edited with the different promoters in the promoter ladder.When the endogenous promoter does not exist, its sequence is unknown, orit has been previously changed in some manner, the individual promotersof the promoter ladder can be inserted in front of the genes ofinterest. These produced cell libraries have individual cells with anindividual promoter of the ladder operably linked to one or more targetgenes in an otherwise identical genetic context. The terminator ladderin question is then associated with a given gene of interest.

The terminator ladder can be used to more generally affect terminationof all or most ORFs in a genome, or a selected subset of the genome,e.g., the ORFs of a kinome or a secretome. The set of target genes canalso be genes known or suspected to be involved in a particular cellularpathway, e.g. a regulatory pathway or signaling pathway. The set oftarget genes can be ORFs related to function, by relation to previouslydemonstrated beneficial edits (previous promoter swaps or previous SNPswaps), by algorithmic selection based on epistatic interactions betweenpreviously generated edits, other selection criteria based on hypothesesregarding beneficial ORF to target, or through random selection. Inspecific embodiments, the target genes can comprise non-protein codinggenes, including non-coding RNAs.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Other equivalent methods, steps and compositionsare intended to be included in the scope of the invention. Efforts havebeen made to ensure accuracy with respect to numbers used (e.g. amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Celsius, and pressure is at or near atmospheric.

Example 1: Assessing the Fitness of a Model Inducible System

Basic components of a model system were assessed. The model systemcomprised E. coli cells transformed with an engine vector, where theengine vector comprised a coding sequence for a MAD nuclease (i.e., MAD4 or MAD 7 nuclease) under the control of the pL temperature induciblepromoter, a chloramphenicol resistance marker, and the λ Redrecombineering system under the control of the pBAD promoter (induced byaddition of arabinose to the growth medium). The E. coli cells were alsotransformed with an editing vector comprising an editingoligonucleotide, which in this model system was a library of editingoligonucleotides each configured to inactivate galK, where successfulediting results in white (versus red) colonies when plated on MacConkeyagar supplemented with galactose as the sole carbon source. In addition,the editing vector comprised a gRNA coding sequence under the control ofthe pL temperature inducible promoter, a carbenicillin resistancemarker, and a sequence to remove, mutate, or otherwise render inactivethe PAM region in the target sequence. FIGS. 11A and 11B depict anexemplary engine vector (FIG. 11A) and editing vector (FIG. 11B) thatmay be employed in the exemplary editing workflows described herein. InFIG. 11A, the exemplary engine vector 1110 (p197) comprises an origin ofreplication 1112, and a promoter 1114 driving expression of the genecoding for the c1857 repressor 1116 which regulates the pL promoter. Afirst pL promoter in this exemplary embodiment drives the transcriptionof the gRNA on the editing vector as described in relation to FIG. 11Bbelow, and a second pL promoter 1118 on the engine vector drivesexpression of the nuclease 1120. The promoter driving nuclease 1120 maybe an inducible or a constitutive promoter; however, the tightestregulation of the nucleic acid-guided nuclease system is achieved byusing an inducible promoter to drive expression of the nuclease as wellas the guide nucleic acid. Again, like inducible promoters and differentinducible promoters may be used to drive the transcription of the guidenucleic acid and the nuclease. The pL promoter can be regulated (e.g.,repressed or induced) by the thermolabile c1857 repressor 1116 which isactive at permissive temperatures (e.g., 30° C.) and inactive at highertemperatures (e.g., 42° C.). The regulation of the pL promoter is shownin more detail in relation to FIG. 1C above.

The engine vector in FIG. 11A further comprises a pBAD promoter 1140driving expression of the components of the λ Red recombineering system1142. The pBAD promoter, like the pL promoter, is an inducible promoter,where the pBAD promoter is regulated (induced) by the addition ofarabinose to the growth medium. Note that in this exemplary bacterialediting system, a recombineering system such as the λ Red recombineeringsystem is provided as a component of the nucleic acid-guided nucleaseediting system to repair the DNA breaks that occur during editing. Insome embodiments, however, the cells to be edited may already comprise arecombineering system (e.g., episomally, integrated into the cellulargenome, or naturally). Also, although the λ Red recombineering system isexemplified here, it should be understood that other recombineeringsystems may be employed. Further, cells—such as yeast, plant and animalcells—do not require a recombineering system equivalent to the λ Redrecombineering system to repair the DNA breaks that result from editing.Thus the nucleic acid-guided nuclease editing components for, e.g.,yeast, plant and animal cells do not need to include a heterologousrecombineering system. Finally, exemplary engine vector 1110 comprises apromoter 1150 driving expression of a chloramphenicol selectable marker1152. Additionally, the engine vector and all other vectors orconstructs used in the disclosed method comprise appropriate controlelements (e.g., polyadenylation signals, enhancers) operably-linked tothe nucleic acid-guided nuclease editing system components.

FIG. 11B depicts an exemplary editing vector 1130 (pLFORGE). Editingvector 1130 comprises an origin of replication 1132, and a pL promoter1134 driving expression of an editing cassette 1136. The editingcassette comprises a coding sequence for a gRNA 1144, a spacer 1146, anda donor DNA 1158 (comprising homology arms flanking a desired edit to bemade to the target sequence). Editing vector 1130 also comprises apromoter 1154 driving expression of a carbenicillin selectable marker1156. Note that the pL inducible promoter 1134 is encoded on the vectorbackbone and is not part of the editing cassette 1136; however, in otherembodiments the inducible promoter driving transcription of the gRNA maybe part of the editing cassette. FIGS. 11A and 11B depict exemplaryengine and editing vectors, respectively, but it should be recognized byone of ordinary skill in the art given the guidance of the presentdescription that all elements of the nucleic acid-guided nucleaseediting system may be contained on a single plasmid, or the elementsshown on the engine and editing vectors of FIGS. 11A and 11B may resideon a different vector than shown. For example, the pBAD promoter and λRed recombineering system may be contained on the editing vector ratherthan the engine vector; likewise, the gene for the c1857 repressor maybe contained on the editing vector rather than the engine vector.

FIG. 12 is a bar graph showing the transformation efficiencies observedfor galK gRNA targeting cassettes under a variety of promoters.Transformation efficiency of uninduced promoters provides a proxy forpromoter leakiness. If there is high basal expression of gRNA (e.g.,“leakiness”), there will be low CFU. If there is a low basal expressionof gRNA (e.g., tight regulation), there will be high CFU. The non-targetgRNAs experiment was performed with a pool of cassettes designed totarget inactive PAM sequences that are unable to promote gRNA andnuclease binding and cleavage. For each of the promoters depicted (pL,pBAD, and sigma32) a cassette targeting galK with a TTTC PAM and aspacer configured to make a stop codon insertion at amino acid D70 wascloned downstream of the promoter. Plasmid concentrations werenormalized to 50 ng/pL and transformed in equal cell volumes. CFU fromserial dilution platings were used to derive the transformationefficiencies expressed as CFU/n. The J23119 promoter is a constitutivepromoter, the pL promoter is inducible by high temperature, the pBADpromoter is induced by the presence of arabinose in the growth medium,and the sigma32 promoter is induced in stationary phase cells. For theMAD7 nuclease, the sigma32 promoter shows leaky expression, while boththe pL and pBAD promoters are tightly repressed.

FIG. 13 is a graph showing the effects of thermal induction on cellviability. In this experiment, a pool of cassettes were designed totarget an inactive PAM sequence. Cells were diluted 1/50 v:v intomicrotiter plates containing 200 μL LB+chlor/carb. Diluted cultures wereinduced for 1 hour at the indicated temperatures before shifting back to30° C. with continuous shaking in an Infinite M Nano+ plate reader. Tocorresponds to the time of the induction step and growth was monitoredby measuring the OD600 at 10 minute intervals. Note that hightemperature (42° C.) induction has no impact on cell viability or growthrate.

FIG. 14 shows the results of pooled induction experiments using a singlesequence verified editing cassette that targets the galK locus andintroduces a stop codon at amino acid D70. Following a three-houroutgrowth, cells were either induced at 42° C. for 15 minutes orretained at 30° C. prior to serial dilution plating. CFU/mL wascalculated based on the plating volumes. For reference the dashed linein FIG. 14 shows the typical CFU obtained in an equivalent experimentusing the constitutive J23119 promoter (data not shown). As can be seen,the absence of induction (i.e., the absence of editing) enables highefficiency transformation (10-100 fold) due to the lack of toxic dsDNAbreaks.

FIG. 15 shows the growth profiles of randomly picked variants from asilent PAM mutation (SPM) library. This 500-member library targetsregions located across the entire E. coli organism and integratessynonymous mutations that have no expected fitness effects. Colonieswere picked from agar plates of uninduced transformed cells. Cells werepicked from an agar plate and grown up in 200 μL LB+chlor/carb overnightin a 96-well microtiter plate format. 10 μL of the well content of theparent microtiter plate was then transferred to two replica daughtermicrotiter plates that received either no induction (top) or gRNA andnuclease induction (via the pL inducible promoter) for 1 hour at 42° C.(bottom). The well maps show the relative OD at 6 hours. The insertsshow examples of the full growth curves for the indicated wells as areference. The replica wells represent growth observed from the samecassette design with or without gRNA induction. While the majority ofthe wells for the no-induction plate show normal growth profiles, theinduced plate shows that a large fraction of the gRNA designs are stillactive when induced, indicated by a large lag phase before the cellsreach exponential growth. That is, the actively-editing cells havereduced viability due to DNA damage such that many in the colonies dieoff, and those edited cells that do survive grow slowly to begin with asthe cellular machinery works to repair the edit. This characteristic ofedited cells can be exploited to screen for active editing in ahigh-throughput manner.

Example 2: Editing Cassette and Backbone Amplification and Assembly

Editing Cassette Preparation:

5 nM oligonucleotides synthesized on a chip were amplified using Q5polymerase in 50 μL volumes. The PCR conditions were 95° C. for 1minute; 8 rounds of 95° C. for 30 seconds/60° C. for 30 seconds/72° C.for 2.5 minutes; with a final hold at 72° C. for 5 minutes. Followingamplification, the PCR products were subjected to SPRI cleanup, where 30μL SPRI mix was added to the 50 μL PCR reactions and incubated for 2minutes. The tubes were subjected to a magnetic field for 2 minutes, theliquid was removed, and the beads were washed 2× with 80% ethanol,allowing 1 minute between washes. After the final wash, the beads wereallowed to dry for 2 minutes, 50 μL 0.5×TE pH 8.0 was added to thetubes, and the beads were vortexed to mix. The slurry was incubated atroom temperature for 2 minutes, then subjected to the magnetic field for2 minutes. The eluate was removed and the DNA quantified.

Following quantification, a second amplification procedure was carriedout using a dilution of the eluate from the SPRI cleanup. PCR wasperformed under the following conditions: 95° C. for 1 minute; 18 roundsof 95° C. for 30 seconds/72° C. for 2.5 minutes; with a final hold at72° C. for 5 minutes. Amplicons were checked on a 2% agarose gel andpools with the cleanest output(s) were identified. Amplificationproducts appearing to have heterodimers or chimeras were not used.

Backbone Preparation:

A 10-fold serial dilution series of purified backbone was performed, andeach of the diluted backbone series was amplified under the followingconditions: 95° C. for 1 minute; then 30 rounds of 95° C. for 30seconds/60° C. for 1.5 minutes/72° C. for 2.5 minutes; with a final holdat 72° C. for 5 minutes. After amplification, the amplified backbone wassubjected to SPRI cleanup as described above in relation to thecassettes. The backbone was eluted into 100 μL ddH₂O and quantifiedbefore Gibson Assembly®.

Gibson Assembly:

150 ng backbone DNA was combined with 100 ng cassette DNA. An equalvolume of 2× Gibson Master Mix was added, and the reaction was incubatedfor 45 minutes at 50° C. After assembly, the assembled backbone andcassettes were subjected to SPRI cleanup, as described above.

Example 3: Transformation of Editing Vector Library into E Cloni®

Transformation:

20 μL of the prepared editing vector Gibson Assembly reaction was addedto 30 μL chilled water along with 10 μL E cloni® (Lucigen, Middleton,Wis.) supreme competent cells. An aliquot of the transformed cells werespot plated to check the transformation efficiency, where >100× coveragewas required to continue. The transformed E cloni® cells were outgrownin 25 mL SOB+100 μg/mL carbenicillin (carb). Glycerol stocks weregenerated from the saturated culture by adding 500 μL 50% glycerol to1000 μL saturated overnight culture. The stocks were frozen at −80° C.This step is optional, providing a ready stock of the cloned editinglibrary. Alternatively, Gibson or another assembly of the editingcassettes and the vector backbone can be performed before each editingexperiment.

Example 4: Preparation of Competent Cells

A 1 mL aliquot of a freshly-grown overnight culture of EC1 cellstransformed with the engine vector was added to a 250 mL flaskcontaining 100 mL LB/SOB+25 μg/mL chlor medium. The cells were grown to0.4-0.7 OD, and cell growth was halted by transferring the culture toice for 10 minutes. The cells were pelleted at 8000×g in a JA-18 rotorfor 5 minutes, washed 3× with 50 mL ice cold ddH₂O or 10% glycerol, andpelleted at 8000×g in JA-18 rotor for 5 minutes. The washed cells wereresuspended in 5 mL ice cold 10% glycerol and aliquoted into 200 μLportions. Optionally at this point the glycerol stocks could be storedat −80° C. for later use.

Example 5: Creation of New Cell Line Transformed with Engine Vector

Transformation:

1 μL of the engine vector DNA (comprising a coding sequence for MAD7nuclease under the control of the pL inducible promoter, achloramphenicol resistance gene, and the λ Red recombineering system)was added to 50 μL EC1 strain E. coli cells. The transformed cells wereplated on LB plates with 25 μg/mL chloramphenicol (chlor) and incubatedovernight to accumulate clonal (or substantially clonal) isolates. Thenext day, a colony was picked, grown overnight in LB+25 μg/mL chlor, andglycerol stocks were prepared from the saturated overnight culture byadding 500 μL 50% glycerol to 1000 μL culture. The stocks of EC1comprising the engine vector were frozen at −80° C.

Example 6: High Throughput Clonal Editing

The following protocols address cell growth/survival bias due to dsDNAbreaks:

Transformation:

100 ng of the editing vector cloned library or editing vector GibsonAssembly® reaction was transformed by electroporation into 100 μLcompetent EC1 cells containing the engine vector. The electroporator wasset to 2400 V in 2 mm cuvette. Following transformation, the cells wereallowed to recover for 3 hours in SOB medium. A 10-fold dilution seriesof recovered cells (in H₂O) was spot plated and the resulting CFUcounts/dilution ratios were used to calculate transformation efficiency.

Plating and Colony Arraying:

100 μL of the appropriate dilution was plated on LB medium+25 μg/mLchlor and grown at 30° C. overnight. Colonies were picked and grownovernight to saturation at 30° C. in 96-well microtiter plates.

Induced Cutting, Editing, and Edit Validation:

A replicator was used to transfer colonies in wells from the overnightgrowth to 200 μL fresh SOB+1% arabinose in replicate 96-well microtiterplates. The replicator transfers approximately 1-2 μL per well resultingin a 100-fold dilution for outgrowth. The microtiter plates wereincubated at 250 rpm shaking for 3-4 hours to allow the cells to reachmid-log phase. The plates were then transferred to a static incubator at42° C. and incubated for 2 hours, then placed in a 30° C. incubator for1 hour to recover. At this point, the replicate cells were ready forgenomic prep and were validated via sequencing analysis. As described inrelation to FIG. 2B, additional replica plates of fresh SOB withoutarabinose may be used to enable functional deconvolution of inactive cutdesigns from the population.

Example 7: Rearray and Pooled Editing Using Inducible gRNAs

Transformation:

100 ng of the editing vector cloned library or Gibson Assembly® reactionwas transformed by electroporation into 100 μL competent EC1 cellscontaining the engine vector. The electroporator was set to 2400 V in 2mm cuvette. Following transformation, the cells were allowed to recoverfor 3 hours in SOB medium. A 10-fold dilution series of recovered cells(in H₂O) was spot plated and the resulting CFU counts/dilution ratioswere used to calculate transformation efficiency.

Plating and Colony Arraying:

100 μL of the appropriate dilution was plated on LB medium+25 μg/mLchlor and grown at 30° C. overnight. Colonies were picked and grownovernight to saturation in 1 mL SOB+25 μg/mL chlor/100 μg/mL carb at 30°C. in 96-well microtiter plates.

Identification of Active Cassettes and Rearray:

A replicator was used to transfer colonies in wells from the overnightgrowth to 200 μL fresh SOB without arabinose in replicate 96-wellmicrotiter plates. The replicator transfers approximately 1-2 μL perwell resulting in a 100-fold dilution for outgrowth. The replicatemicrotiter plates were incubated at 250 rpm shaking for 3-4 hours toallow the cells to reach mid-log growth. The plates were thentransferred to a static incubator at 42° C. and incubated for 2 hours,then placed in a 30° C. incubator for 1 hour to recover. The replicatorwas then used to transfer cells from the replicate microtiter plates toagar plates with LB medium+25 μg/mL chlor/100 μg/mL carb and allowed togrow overnight. Wells with active gRNAs were identified and 50 μL ofcells from these colonies from the original 96-well microtiter platewere re-arrayed into “functionally-corrected” plates (e.g., the cellsidentified as having functional gRNAs were transferred (cherry picked)into a 96-well plate).

Pooled Editing Using Pooled Cassettes Identified as Active:

100 μL of cells identified as having functional gRNAs were transferredinto 5 mL SOB medium and grown until the culture was saturated. 1%arabinose was added to the tube and the tube was incubated for 2 hoursat 30° C. Cutting/editing was induced by transferring the tube to a 42°C. shaking water bath for 2 hours. The tube was then removed from thewater bath and allowed to recover for 2 hours at 30° C. in a 250 rpmshaking incubator. A diluted culture (approximately 10⁻⁴ to 10⁻⁵) wasplated on LB medium+25 μg/mL chlor/100 μg/mL carb resulting in isolatedclonal colonies. Editing was assessed/validated by targeted or wholegenome sequencing.

Example 8: Error Correction: Re-Array and Cloning

Transformation:

100 ng of the editing vector cloned library or Gibson Assembly® reactionwas transformed by electroporation into 100 μL competent EC1 cellscontaining the engine vector. The electroporator was set to 2400 V in 2mm cuvette. Following transformation, the cells were allowed to recoverfor 3 hours in SOB medium. A 10-fold dilution series of recovered cells(in H₂O) was spot plated and the resulting CFU counts/dilution ratioswere used to calculate transformation efficiency.

Plating and Colony Arraying:

100 μL of the appropriate dilution was plated on LB medium+25 μg/mLchlor and grown at 30° C. overnight. Colonies were picked and grownovernight to saturation in 1 mL SOB+25 μg/mL chlor and 100 μg/mL carb at30° C. in 96-well microtiter plates.

Identification of Active Cassettes and Rearray:

A replicator was used to transfer colonies in wells from the overnightgrowth to 200 μL fresh SOB without arabinose in replicate 96-wellmicrotiter plates. The replicator transfers approximately 1-2 μL perwell resulting in a 100-fold dilution for outgrowth. The replicatemicrotiter plates were incubated at 250 rpm shaking for 3-4 hours toallow the cells to reach mid-log growth. The plates were thentransferred to a static incubator at 42° C. and incubated for 2 hours,then placed in a 30° C. incubator for 1 hour to recover. The replicatorwas then used to transfer cells from the replicate microtiter plates toplates with LB medium+25 μg/mL chlor/100 μg/mL carb and allowed to growovernight. Wells with active gRNAs were identified and 50 μL of cellsfrom these colonies from the original 96-well microtiter plate werere-arrayed into “functionally-corrected” plates (e.g., the cellsidentified as having functional gRNAs were transferred into a 96-wellplate).

Library Re-Amplification, Cloning, and Validation:

Cells identified as having functional gRNAs were pooled and DNA wasextracted and isolated. Serial dilutions were made of the isolated DNA,and amplification was conducted with primers designed to amplify theediting cassettes. PCR was performed under the following conditions: 95°C. for 1 minute; 18 rounds of 95° C. for 30 seconds/60° C. for 30seconds/72° C. for 2.5 minutes; with a final hold at 72° C. for 5minutes. Amplicons were checked on a 1% agarose gel and pools with thecleanest output(s) were identified. The amplified cassettes weremini-prepped and eluted into 50 μL ddH₂O. Next, a Gibson Assembly®reaction containing 150 ng backbone DNA with 100 ng cassette inserts wasperformed. An equal volume 2× Master Mix was added to the backbone andinsert, and the reaction was incubated for 45 min @ 50° C., thendialyzed for 30 min in sitting droplet with 0.25 μm filter disc. 100 ngof the Gibson Assembly® reaction was transformed by electroporation intocompetent EC1 cells containing the engine vector as described above.Following transformation, the cells were allowed to recover for 3 hoursin SOB medium. A 10-fold dilution series of recovered cells (in H₂O) wasspot plated and the resulting CFU counts/dilution ratios were used tocalculate transformation efficiency. 100 μL of an appropriate dilutionof cells were plated on LB medium+25 μg/mL chlor and grown at 30° C.overnight. Editing was assessed/validated by, e.g., sequencing.

Example 9: Enrichment of Editing Cells by Growth Lag Identification

Transformation:

100 ng of the editing vector cloned library or Gibson Assembly® reactionwas transformed by electroporation into 100 μL competent EC1 cellscontaining the engine vector. The electroporator was set to 2400 V in 2mm cuvette. Following transformation, the cells were allowed to recoverfor 3 hours in SOB medium. A 10-fold dilution series of recovered cells(in H₂O) was spot plated and the resulting CFU counts/dilution ratioswere used to calculate transformation efficiency.

Plating and Colony Arraying:

100 μL of the appropriate dilution was plated on LB agar medium+25 μg/mLchlor and +arabinose and grown at 30° C. for 6-8 hours. Alternatively,the cells may be grown in liquid culture in LB medium+25 μg/mL chlor at30° C. to saturation and diluted to the appropriate concentration beforeplating on LB agar medium+25 μg/mL chlor+arabinose and grown at 30° C.for 6-8 hours. Following the 6-8 hour growth, the temperature of theplate was adjusted to 42° C. and the plates were incubated for twohours. The temperature was then adjusted back to 30° C. and the cellswere allowed to recover overnight.

Edited Cell Identification:

Small-size colonies were identified. The small colony-size phenotypeindicates cell viability was compromised during the induced-editingprocedure. Efficient recovery of edited cells from the initial pool wasaccomplished by identifying and picking small colonies and arrayingcells from these small colonies onto a 96-well plate to create a libraryof edited cells, or the cells from the colonies were pooled generatinghighly-edited cell populations for recursive editing. Editing wasassessed/validated by sequencing. It was found that 85% of the smallcolonies were edited cells.

Example 10: Standard Plating Protocol Control

This protocol describes a standard plating protocol for enriching fornucleic acid-guided nuclease editing of bacterial cells by singulationor substantial singulation, growth, editing, and normalization. Thisprotocol was used to leverage the inducible system for both the nucleaseand gRNAs to allow for a phenotypic difference in colonies. From theresulting agar plates, it was possible to select edited cells with ahigh degree (˜80%) of confidence. Though clearly this protocol can beemployed for enriching for edited cells, in the experiments describedherein this “standard plating protocol,” or “SPP,” was used to compareefficiencies of singulation or substantial singulation, editing, andnormalization with the solid wall device. Materials: Outside of standardmolecular biology tools, the following will be necessary:

TABLE 1 Product Vendor SOB Teknova LB Teknova LB agar plate with Teknovachloramphenicol/carbenicillin and 1% arabinose

Protocol: Inputs for this protocol are frozen electrocompetent cells andpurified nucleic acid assembly product. Immediately afterelectroporation, the cell/DNA mixture was transferred to a culture tubecontaining 2.7 mL of SOB medium. Preparing 2.7 mL aliquots in 14 mLculture tubes prior to electroporation allowed for a faster recovery ofcells from the electroporation cuvette; the final volume of the recoverywas 3 mL. All culture tubes were placed into a shaking incubator set to250 rpm and 30° C. for three hours. While the cultures were recovering,the necessary number of LB agar plates with chloramphenicol andcarbenicillin+1% arabinose were removed from the refrigerator and warmedto room temperature. Multiple dilutions were used for each plating so asto have countable and isolated colonies on the plates. Platingsuggestions:

TABLE 2 Dilution(s) Sequencing type suggested Volume to plate SinglePlex10⁻¹ through 10⁻³ 300 μL Amplicon None 300 μL (= 1/10^(th) recovery)

After three hours, the culture tubes were removed from the shakingincubator. First, plating for amplicon sequencing was performed byfollowing the above table. Plating beads were used to evenly distributethe culture over the agar. The beads were removed from the plate and theplate was allowed to dry uncovered in a laminar flow cabinet. While theplates were drying, the remaining culture was used to perform serialdilutions, where the standard dilutions were 50 μL of culture into 450μL of sterile, 0.8% NaCl. The plate/tubes used for these dilutions (aswell as the original culture) were maintained at 4° C. in caseadditional dilutions were needed to be performed based on colony counts.Plating for SinglePlex was performed according to the Table 2.Additional or fewer dilutions may be used based on library/competentcell knowledge. The cultures were evenly spread across the agar usingsterile, plating beads. The beads were then removed from the plate andthe plates were allowed to dry uncovered in the laminar flow cabinet.While the plates were drying, an incubator was programmed according tothe following settings: 30° C. for 9 hours→42° C. for 2 hours→30° C. for9 hours. The agar plates were placed in the pre-set incubator, and afterthe temperature cycling was complete (˜21 hours), the agar plates wereremoved from the incubator. Induction of editing was successful asindicated by the size differences in the resulting cell colonies.

Example 11: Singulation and Culture of E. Coli in a Solid Wall Device

Electrocompetent E. coli cells were transformed with a cloned library,an isothermal assembled library, or a process control sgRNA plasmid(escapee surrogate) as described in Examples 2-5 above. The E. colistrain carried the appropriate endonuclease and lambda red componentsand editing induction system (e.g., on an engine plasmid or integratedinto the bacterial genome or a combination). Transformations routinelyused 150 ng of plasmid DNA (or Gibson Assembly reactions) with 150 ng ofpL sgRNA backbone DNA. Following electroporation, the cells were allowedto recover in 3 mL SOB and incubated at 30° C. with shaking for 3 hours.In parallel with processing samples through the solid wall device,samples were also processed with the solid plating protocol (see Example10 above), so as to compare “normalization” in the sold wall device withthe standard benchtop process. Immediately before cells the cells wereintroduced to the permeable-bottom solid wall device, the 0.2 μm filterforming the bottom of the microwells was treated with a 0.1% TWEEN(polysorbate or, IUPAC, polyoxyethylene (20) sorbitan monolaurate)solution to effect proper spreading/distribution of the cells into themicrowells of the solid wall device. The filters were placed into aSwinnex Filter Holder (47 mm, Millipore®, SX0004700) and 3 mL of asolution with 0.85% NaCl, and 0.1% TWEEN (polysorbate or, IUPAC,polyoxyethylene (20) sorbitan monolaurate) was pulled through the solidwall device and filter through using a vacuum. Different TWEEN(polysorbate or, IUPAC, polyoxyethylene (20) sorbitan monolaurate)concentrations were evaluated, and it was determined that for a 47 mmdiameter solid wall device with a 0.2 μM filter forming the bottom ofthe microwells, a pre-treatment of the solid wall device+filter with0.1% TWEEN (polysorbate or, IUPAC, polyoxyethylene (20) sorbitanmonolaurate) was preferred (data not shown).

After the 3-hour recovery in SOB, the transformed cells were diluted anda 3 mL volume of the diluted cells was processed through the TWEEN(polysorbate or, IUPAC, polyoxyethylene (20) sorbitanmonolaurate)-treated solid wall device and filter, again using a vacuum.The number of successfully transformed cells was expected to beapproximately 1.0E+06 to 1.0E+08, with the goal of loading approximately10,000 transformed cells into the current 47 mm permeable-bottom solidwall device (having ˜30,000 wells). Serial dilutions of 10⁻¹, 10⁻², and10⁻³ were prepared, then 100 μL volumes of each of these dilutions werecombined with 3 mL 0.85% NaCl, and the samples were loaded onto solidwall devices. Each permeable-bottom solid wall device was then removedfrom the Swinnex filter holder and transferred to an LB agar platecontaining carbenicillin (100 μg/mL), chloramphenicol (25 μg/mL) andarabinose (1% final concentration). The solid wall devices were placedmetal side “up,” so that the permeable-bottom membrane was touching thesurface of the agar such that the nutrients from the plate could travelup through the filter “bottom” of the wells. The solid wall devices onthe LB agar plates were incubated for 9 hours at 30° C., at 42° C. for 2hours, then returned to incubation at 30° C., for 12-16 hour, and, inanother experiment for 36-40 hours.

At the end of the incubation the perforated disks and filters (stillassembled) were removed from the supporting nutrient source (in thiscase an agar plate) and were photographed with a focused,“transilluminating” light source so that the number and distribution ofloaded microwells on the solid wall device could be assessed (data notshown). To retrieve cells from the permeable-bottom solid wall device,the filter was transferred to a labeled sterile 100 mm petri dish whichcontained 15 mL of sterile 0.85% NaCl, then the petri dish was placed ina shaking incubator set to 30° C./80 RPM to gently remove the cells fromthe filter and resuspend the cells in the 0.85% NaCl. The cells wereallowed cells to shake for 15 minutes, then were transferred to asterile tube, e.g., a 50 mL conical centrifuge tube. The OD600 of thecell suspension was measured and at this point, the cells can beprocessed in different ways depending on the purpose of the study. Forexample, if the plasmids or libraries are designed to target a sugarmetabolism pathway gene such as galK, then the resuspended cells can bespread onto MacConkey agar plates containing galactose (1% finalconcentration) and the appropriate antibiotics. On this differentialmedium, colonies that are the result of successfully-edited cells areexpected to be phenotypically white in color, whereas unedited coloniesare red in color. This red/white phenotype can then be used to assessthe percentage of edited cells and the extent of normalization of editedand unedited cells. The results of one experiment are shown below inTable 3. In all replicates, the transformed cells were allowed to growin the solid wall devices for 9 hours at 30° C., 2 hours at 42° C., andovernight at 30° C.

TABLE 3 TWEEN (polysorbate or IUPAC, polyoxytheylene (20) sorbitanDilution Red White monolaurate) counted colonies colonies % edit NoTWEEN 10⁻⁴ 72 5 6% No TWEEN 10⁻⁴ 89 3 3% No TWEEN 10⁻³ 64 5 7%Pre-treatment TWEEN 10⁻⁴ 71 5 7% Pre-treatment TWEEN 10⁻³ 443 29 6%Pre-treatment TWEEN 10⁻³ 149 12 7% Pre-treatment TWEEN 10⁻³ 83 21 20% Pre-treatment TWEEN 10⁻² 318 112 26%  Pre-treatment TWEEN + 10⁻³ 163 2513%  TWEEN in cell loading buffer Pre-treatment TWEEN + 10⁻⁴ 132 10 7%TWEEN in cell loading buffer Pre-treatment TWEEN + 10⁻⁴ 31 9 23%  TWEENin cell loading buffer Pre-treatment TWEEN + 10⁻³ 147 18 10.9%   TWEENin cell loading buffer Pre-treatment TWEEN + 10⁻² 720 150 17%  TWEEN incell loading buffer Pre-treatment TWEEN + 10⁻³ 55 15 21%  TWEEN in cellloading buffer

FIG. 16 is a graph showing the extent of normalization of cells (%edited cells) for different dilutions of transformed cells, and notreatment with TWEEN (polysorbate or IUPAC, polyoxytheylene (20)sorbitan monolaurate) vs. pre-treatment with TWEEN (polysorbate or,IUPAC, polyoxyethylene (20) sorbitan monolaurate) vs. pre-treatment withTWEEN (polysorbate or, IUPAC, polyoxyethylene (20) sorbitanmonolaurate)+TWEEN (polysorbate or, IUPAC, polyoxyethylene (20) sorbitanmonolaurate) in the buffer when loading the cells into the microwells ofthe solid wall device. A standard plating protocol (SPP) was conductedin parallel with the solid wall singulation experiments as a benchmark(first bar on the left in the graph). Note that the percentage of editsfor the standard plating protocol was approximately 27.5%, and thepercentage of edits for two replicates of the 10⁻³ dilution of cellswith pre-treatment with TWEEN (polysorbate or, IUPAC, polyoxyethylene(20) sorbitan monolaurate) was approximately 20% and 26%, respectively.

Example 12: Singulation of Yeast Colonies in a Solid Wall Device

Electrocompetent yeast cells were transformed with a cloned library, anisothermal assembled library, or a process control sgRNA plasmid(escapee surrogate) as described in Examples 2-5 above. ElectrocompetentSaccharomyces cerevisiae cells were prepared as follows: The afternoonbefore transformation was to occur, 10 mL of YPAD was inoculated withthe selected Saccharomyces cerevisiae strain. The culture was shaken at250 RPM and 30° C. overnight. The next day, 100 mL of YPAD was added toa 250-mL baffled flask and inoculated with the overnight culture (around2 mL of overnight culture) until the OD600 reading reached 0.3+/−0.05.The culture was placed in the 30° C. incubator shaking at 250 RPM andallowed to grow for 4-5 hours, with the OD checked every hour. When theculture reached an OD600 of approximately 1.5, 50 mL volumes were pouredinto two 50-mL conical vials, then centrifuged at 4300 RPM for 2 minutesat room temperature. The supernatant was removed from all 50 ml conicaltubes, while avoiding disturbing the cell pellet. 50 mL of a LithiumAcetate/Dithiothreitol solution was added to each conical tube and thepellet was gently resuspended. Both suspensions were transferred to a250 mL flask and placed in the shaker; then shaken at 30° C. and 200 RPMfor 30 minutes. After incubation was complete, the suspension wastransferred to two 50-mL conical vials. The suspensions then werecentrifuged at 4300 RPM for 3 minutes, then the supernatant wasdiscarded.

Following the Lithium Acetate/Dithiothreitol treatment step, coldliquids were used and the cells were kept on ice until electroporation.50 mL of 1 M sorbitol was added and the pellet was resuspended, thencentrifuged at 4300 RPM, 3 minutes, 4° C., after which the supernatantwas discarded. The 1 M sorbitol wash was repeated twice for a total ofthree washes. 50 μL of 1 M sorbitol was added to one pellet, cells wereresuspended, then transferred to the other tube to suspend the secondpellet. The volume of the cell suspension was measured and brought to 1mL with cold 1 M sorbitol. At this point the cells were electrocompetentand could be transformed with a cloned library, an isothermal assembledlibrary, or process control sgRNA plasmids. In brief, a required numberof 2-mm gap electroporation cuvettes were prepared by labeling thecuvettes and then chilling on ice. The appropriate plasmid—or DNAmixture—was added to each corresponding cuvette and placed back on ice.100 μL of electrocompetent cells was transferred to each labelledcuvette, and each sample was electroporated using appropriateelectroporator conditions. 900 μL of room temperature YPAD Sorbitolmedia was then added to each cuvette. The cell suspension wastransferred to a 14 mL culture tube and then shaken at 30° C., 250 RPMfor 3 hours. After a 3 hr recovery, 9 mL of YPAD containing theappropriate antibiotic, e.g., geneticin or Hygromycin B, was added.

At this point the transformed cells were processed in parallel in thesolid wall device and the standard plating protocol (see Example 10above), so as to compare “normalization” in the sold wall device withthe standard benchtop process. Immediately before cells the cells wereintroduced to the permeable-bottom solid wall device, the 0.45 μM filterforming the bottom of the microwells was treated with a 0.1% TWEEN(polysorbate or, IUPAC, polyoxyethylene (20) sorbitan monolaurate)solution to effect proper spreading/distribution of the cells into themicrowells of the solid wall device. The filters were placed into aSwinnex Filter Holder (47 mm, Millipore®, SX0004700) and 3 mL of asolution with 0.85% NaCl and 0.1% TWEEN (polysorbate or, IUPAC,polyoxyethylene (20) sorbitan monolaurate) was pulled through the solidwall device and filter through using a vacuum. Different TWEEN(polysorbate or, IUPAC, polyoxyethylene (20) sorbitan monolaurate)concentrations were evaluated, and it was determined that for a 47 mmdiameter solid wall device with a 0.45 μM filter forming the bottom ofthe microwells, a pre-treatment of the solid wall device+filter with0.1% TWEEN (polysorbate or, IUPAC, polyoxyethylene (20) sorbitanmonolaurate) was preferred (data not shown).

After the 3-hour recovery in SOB, the transformed cells were diluted anda 3 mL volume of the diluted cells was processed through the TWEEN(polysorbate or, IUPAC, polyoxyethylene (20) sorbitanmonolaurate)-treated solid wall device and filter, again using a vacuum.The number of successfully transformed cells was expected to beapproximately 1.0E+06 to 1.0E+08, with the goal of loading approximately10,000 transformed cells into the current 47 mm permeable-bottom solidwall device (having ˜30,000 wells). Serial dilutions of 10⁻¹, 10⁻², and10⁻³ were prepared, then 100 μL volumes of each of these dilutions werecombined with 3 mL 0.85% NaCl, and the samples were loaded onto solidwall devices. Each permeable-bottom solid wall device was then removedfrom the Swinnex filter holder and transferred to an LB agar platecontaining carbenicillin (100 μg/mL), chloramphenicol (25 μg/mL) andarabinose (1% final concentration). The solid wall devices were placedmetal side “up,” so that the permeable-bottom membrane was touching thesurface of the agar such that the nutrients from the plate could travelup through the filter “bottom” of the wells. The solid wall devices onthe YPD agar plates were incubated for 2-3 days at 30° C.

At the end of the incubation, the perforated disks and filters (stillassembled) were removed from the supporting nutrient source (in thiscase an agar plate) and were photographed with a focused,“transilluminating” light source so that the number and distribution ofloaded microwells on the solid wall device could be assessed (see FIGS.17A and 17B). To retrieve cells from the permeable-bottom solid walldevice, the filter was transferred to a labeled sterile 100 mm petridish which contained 15 mL of sterile 0.85% NaCl, then the petri dishwas placed in a shaking incubator set to 30° C./80 RPM to gently removethe cells from the filter and resuspend the cells in the 0.85% NaCl. Thecells were allowed to shake for 15 minutes, then were transferred to asterile tube, e.g., a 50 mL conical centrifuge tube. The OD600 of thecell suspension was measured; at this point the cells can be processedin different ways depending on the purpose of the study. For example, ifan ADE2 stop codon mutagenesis library is used, successfully-editedcells should result in colonies with a red color phenotype when theresuspended cells are spread onto YPD agar plates and allowed to growfor 4-7 days. This phenotypic difference allows for a quantification ofpercentage of edited cells and the extent of normalization of edited andunedited cells.

Example 13: Singulation, Growth and Editing of E. coli in 200K SWIIN

Singleplex automated genomic editing using MAD7 nuclease, a library with94 different edits in a single gene (yagP) and employing a 200Ksingulation device such as those exemplified in FIGS. 4F-4R wassuccessfully performed. The engine vector used was substantially similarto that depicted in FIG. 11A (with MAD7 under the control of the pLinducible promoter), and the editing vector used was substantiallysimilar to that depicted in FIG. 11B—including the editing cassettebeing under the control of the pL inducible promoter, and the λ Redrecombineering system under control of the pBAD inducible promoterpBAD—with the exception that the editing cassette comprises the 94 yagPgene edits (donor DNAs) and the appropriate corresponding gRNAs. TwoSWIIN workflows were compared, and further were benchmarked against thestandard plating protocol (see Example 7). The SWIIN protocols differentfrom one another that in one set of replicates LB medium containingarabinose was used to distribute the cells in the SWIIN (arabinose wasused to induce the λ Red recombineering system (which allows for repairof double-strand breaks in E. coli that are created during editing), andin the other set of replicates SOB medium without arabinose was used todistribute the cells in the SWIIN and for initial growth, with mediumexchange performed to replace the SOB medium without arabinose with SOBmedium with arabinose. Approximately 70K cells were loaded into the 200KSWIIN.

In all protocols (standard plating, LB-SWIIN, and SOB-SWIIN), the cellswere allowed to grow at 30° C. for 9 hours and editing was induced byraising the temperature to 42° C. for 2.5 hours, then the temperaturewas returned to 30° C. and the cells were grown overnight. The resultsof this experiment are shown in FIG. 18 and in Table 4 below. Note thatsimilar editing performance was observed with the four replicates of thetwo SWIIN workflows, indicating that the performance of SWIIN platingwith and without arabinose in the initial medium is similar. Editingpercentage in the standard plating protocol was approximately 77%, inbulk liquid was approximately 67%, and for the SWIIN replicates rangedfrom approximately 63% to 71%. Note that the percentage of unique editcassettes divided by the total number of edit cassettes was similar foreach protocol.

TABLE 4 SWIIN SWIIN SWIIN SWIIN SOB then SOB then Standard LB/Ara LB/AraSOB/Ara SOB/Ara Plating Rep. A Rep. B Rep. A Rep. B 40006 edit calls/0.777 0.633 0.719 0.663 0.695 identified wells Unique edit 0.49 0.490.43 0.50 0.51 cassettes/total edit cassettes

Example 14: Fully-Automated Singleplex RGN-Directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument such asthat shown in FIGS. 5A-5D. See U.S. Pat. No. 9,982,279, issued 29 May2018 and Ser. No. 10/240,167, issued 9 Apr. 2019.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via Gibson Assembly® into an “editing vector” in an isothermalnucleic acid assembly module included in the automated instrument.lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicatesthat the edit happens at the 172nd residue in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The assembled editing vector andrecombineering-ready, electrocompetent E. Coli cells were transferredinto a transformation module for electroporation. The cells and nucleicacids were combined and allowed to mix for 1 minute, and electroporationwas performed for 30 seconds. The parameters for the poring pulse were:voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1;polarity, +. The parameters for the transfer pulses were: Voltage, 150V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/−.Following electroporation, the cells were transferred to a recoverymodule (another growth module) and allowed to recover in SOC mediumcontaining chloramphenicol. Carbenicillin was added to the medium after1 hour, and the cells were allowed to recover for another 2 hours. Afterrecovery, the cells were held at 4° C. until recovered by the user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example 15: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E. Coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in theisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. Coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

TABLE 5 Valve Status and Pressure SV9- SV10- Description of Manifold/SV1- SV2- SV3- SV4- SV5- SV6- SV7- SV8- PUMP PUMP step Step arm Pump PR1PR2 RR1 RR2 PR3 PR4 PR5 RR4 “P” “V” psi Load SWIIN 1 open 0 0 0 0 0 0 00 0 0 0 0 module on instrument Load 7.5 ml of 2 open 0 0 0 0 0 0 0 0 0 00 0 cell suspension into RR1 and RR3 Fill retentate 3 closed 1 0 0 1 0 00 1 0 0 0 0.5 layer with cell suspension Let cells settle to 4 closed 00 0 0 0 0 0 0 0 0 0 0 the vicinity of the perforated sheet and intomicrowells Drive medium in 5 closed 1 1 1 0 0 1 1 0 0 1 1 −5 theretentate layer across membrane Open manifold 6 open 0 0 0 0 0 0 0 0 0 00 0 Remove excess 7 open 0 0 0 0 0 0 0 0 0 0 0 0 medium from PRs withpipettor Load 7.5 ml 8 open 0 0 0 0 0 0 0 0 0 0 0 0 growth medium intoPR1 and PR3 Close manifold 9 closed 0 0 0 0 0 0 0 0 0 0 0 0 Flushpermeate 10 closed 0 0 0 0 0 0 0 0 0 0 0 0 layer with growth medium fromPR1 to PR2 and PR3 Open manifold 11 open 0 0 0 0 0 0 0 0 0 0 0 0 Removeexcess 12 open 0 0 0 0 0 0 0 1 0 0 0 0.5 medium from PRs with pipettorLoad 7.5 ml 13 open 0 0 0 0 0 0 0 0 0 0 0 0 growth medium into PR1 andPR3 Close manifold 14 closed 0 0 0 0 0 0 1 0 0 1 1 −5 Flush permeate 15closed 0 0 0 0 0 0 0 0 0 0 0 0 layer with growth medium from PR1 to PR2and PR3 to PR4 via gravity Open manifold 16 open 0 0 0 0 0 0 0 0 0 0 0 0Remove excess 17 open 0 0 0 0 0 0 0 0 0 0 0 0 medium from PRs withpipettor Close manifold 18 closed 0 0 0 0 0 0 0 0 0 0 0 0 Incubate at30° C. 19 closed 1 0 0 1 0 0 0 1 0 0 0 0.25 for 4.5 hours Open manifold20 open 0 0 0 0 0 0 0 0 0 0 0 0 Load 7.5 ml 21 open 0 0 0 0 0 0 0 0 0 00 0 growth medium into PR1 and PR3 Close manifold 22 closed 0 0 0 0 0 00 0 0 0 0 0 Incubate at 30° C. 23 closed 1 0 0 1 0 0 0 1 0 0 0 0.25 for4.5 hours Induce at 42° C. 24 closed 1 0 0 1 0 0 0 1 0 0 0 0.25 for 2hours Incubate at 30° C. 25 closed 1 0 0 1 0 0 0 1 0 0 0 0.25 for 9hours Aspirate cells 26 closed 1 0 0 1 0 0 1 0 0 1 1 −5 into RR1 and RR2Open manifold 27 open 1 0 0 0 0 0 0 0 0 0 0 0 Recover cells 28 open 1 00 0 0 0 0 0 0 0 0 0 from RR1 and RR3 with pipettor Remove 29 open 1 0 00 0 0 0 0 0 0 0 0 remaining medium from PR1 and PR2 and PR3 and PR4

TABLE 6 Reservoir Volumes RR1 and RR3 RR2 and RR4 PR1 and PR3 PR2 andPR4 Temperature Description of step Step Initial/Final Initial/FinalInitial/Final Initial/Final (° C.) Load SWIIN module on instrument 1 0/00/0 0/0 0/0 25 Load 7.5 ml of cell suspension into 2   0/7.5 TBD/TBD 0/00/0 25 RR1 and RR3 Fill retentate layer with cell 3 7.5/0   TBD/TBD 0/00/0 25 suspension Let cells settle to the vicinity of the 4 0/0 TBD/TBD0/0 0/0 4 perforated sheet and into microwells Drive medium in theretentate layer 5 0/0 TBD/TBD 0/1 0/1 30 across membrane Open manifold 60/0 TBD/TBD 1/1 1/1 30 Remove excess medium from PRs 7 0/0 TBD/TBD 1/01/0 30 with pipettor Load 7.5 ml growth medium into 8 0/0 TBD/TBD  0/7.5 0/0 30 PR1 and PR3 Close manifold 9 0/0 TBD/TBD 7.5/7.5 0/0 30Flush permeate layer with growth 10 0/0 TBD/TBD  7.5/3.75   0/3.75 30medium from PR1 to PR2 and PR3 Open manifold 11 0/0 TBD/TBD 3.75/3.753.75/3.75 30 Remove excess medium from PRs 12 0/0 TBD/TBD 3.75/0  3.75/0   30 with pipettor Load 7.5 ml growth medium into 13 0/0 TBD/TBD  0/7.5 0/0 30 PR1 and PR3 Close manifold 14 0/0 TBD/TBD 7.5/7.5 0/0 30Flush permeate layer with growth 15 0/0 TBD/TBD  7.5/3.75   0/3.75 30medium from PR1 to PR2 and PR3 to PR4 via gravity Open manifold 16 0/0TBD/TBD 3.75/3.75 3.75/3.75 30 Remove excess medium from PRs 17 0/0TBD/TBD 3.75/0   3.75/0   30 with pipettor Close manifold 18 0/0 TBD/TBD0/0 0/0 30 Incubate at 30° C. for 4.5 hours 19 0/0 TBD/TBD 0/0 0/0 30Open manifold 20 0/0 TBD/TBD 0/0 0/0 30 Load 7.5 ml growth medium into21 0/0 TBD/TBD   0/7.5 0/0 30 PR1 and PR3 Close manifold 22 0/0 TBD/TBD 7.5/3.75   0/3.75 30 Incubate at 30° C. for 4.5 hours 23 0/0 TBD/TBD3.75/3.75 3.75/3.75 30 Induce at 42° C. for 2 hours 24 0/0 TBD/TBD3.75/3.75 3.75/3.75 42 Incubate at 30° C. for 9 hours 25 0/0 TBD/TBD3.75/3.75 3.75/3.75 30 Aspirate cells into RR1 and RR2 26 0/5 TBD/TBD3.75/1.25 3.75/1.25 RT Open manifold 27 5/5 TBD/TBD 1.25/1.25 1.25/1.25RT Recover cells from RR1 and RR3 28 5/0 TBD/TBD 1.25/1.25 1.25/1.25 RTwith pipettor Remove remaining medium from 29 0/0 TBD/TBD 1.25/0  1.25/0   RT PR1 and PR2 and PR3 and PR4

We claim:
 1. A method for enriching edited cells during CRISPR editingcomprising: transforming cells with one or more vectors comprising apromoter driving transcription of a coding sequence for a CRISPRnuclease, an inducible promoter driving transcription of a guide nucleicacid sequence covalently-linked to a DNA donor sequence; diluting thetransformed cells to a cell concentration to substantially singulate thetransformed cells on a substrate; growing the substantially singulatedtransformed cells on the substrate until the substantially singulatedtransformed cells establish colonies; initiating editing by inducing theinducible promoter driving transcription of the guide nucleic acid; andgrowing the edited cells to form colonies of terminal size.
 2. Themethod of claim 1, wherein the inducible promoter is a pL promoter. 3.The method of claim 1, wherein all of the coding sequence for the CRISPRnuclease, guide nucleic acid sequence covalently-linked DNA donorsequence are on the same vector.
 4. The method of claim 1, wherein thecoding sequence for the CRISPR nuclease is on a first vector and theguide nucleic acid sequence and DNA donor sequence are on a secondvector.
 5. The method of claim 1, wherein the DNA donor sequence furthercomprises a PAM-altering sequence.
 6. The method of claim 1, wherein theedited cells are mammalian cells.
 7. The method of claim 1, wherein theedited cells are yeast cells.
 8. The method of claim 1, wherein theedited cells are bacterial cells.
 9. The method of claim 8, wherein theone or more vectors further comprises a recombineering system.
 10. Themethod of claim 1, wherein the substrate is selected from an agar plateand a device with solid walls.
 11. The method of claim 10, wherein thedevice with solid walls is a solid wall isolation, induction, andnormalization (SWIIN) device.
 12. A method for enriching edited cellsduring CRISPR nuclease editing comprising: transforming cells with oneor more vectors comprising a promoter driving transcription of a codingsequence for a CRISPR nuclease, an inducible promoter drivingtranscription of a guide nucleic acid sequence covalently-linked to aDNA donor sequence, and a gene for a selectable marker; diluting thetransformed cells to a cell concentration to substantially singulate thetransformed cells on a solid wall device; growing the substantiallysingulated transformed cells on the substrate until the substantiallysingulated transformed cells establish colonies; initiating editing byinducing the inducible promoter; and growing the edited cells to formcolonies of terminal size.
 13. The method of claim 12, wherein thepromoter driving transcription of the guide nucleic acid is a pLpromoter.
 14. The method of claim 12, wherein the solid wall device is asolid wall isolation, induction, and normalization (SWIIN) device. 15.The method of claim 12, wherein the one or more vectors each comprise aselectable marker and further comprising adding selective agents to thesubstrate to select for the one or more vectors.
 16. The method of claim12, wherein the CRISPR nuclease is a MADzyme nuclease.
 17. The method ofclaim 12, further comprising pooling the terminal-size colonies.
 18. Themethod of claim 17, further comprising subjecting the pooledterminal-size colonies to another round of transformation, dilution,growth, editing, and growth to terminal-size colonies.
 19. The method ofclaim 12, wherein the transformed cells are mammalian cells.
 20. Themethod of claim 12, wherein the transformed cells are bacterial cellsand the one or more vectors further comprise a recombineering system.